Growth factor-induced proliferation of neural precursor cells in vivo

ABSTRACT

A method is described for inducing in vivo proliferation of precursor cells located in mammalian neural tissue by administering to the mammal a fibroblast growth factor and at least one additional growth factor selected from the group consisting of epidermal growth factor, transforming growth factor alpha, and amphiregulin. The method can be used to replace damaged or missing neurons and/or glia. Another method is described for transplanting multipotent neural stem cell progeny into a mammal. The method comprises the steps of administering growth factors to a mammal to induce in vivo proliferation of neural precursor cells, removing the precursor cell progeny from the mammal, culturing the removed cells in vitro in the presence of one or more growth factors that induces multipotent neural stem cell proliferation, and implanting the multipotent neural stem cell progeny into the mammal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/270,412,filed Jul. 5, 1994, now abandoned, which is a continuation of U.S. Ser.No. 07/726,812, filed Jul. 8, 1991, now abandoned; acontinuation-in-part of U.S. Ser. No. 08/385,404, filed Feb. 7, 1995,now abandoned, which is a continuation of U.S. Ser. No. 07/961,813,filed Oct. 16, 1992, now abandoned, which is a continuation-in-part ofU.S. Ser. No. 07/726,812, filed Jul. 8, 1991, now abandoned; acontinuation-in-part of U.S. Ser. No. 08/359,945, filed Dec. 20, 1994,no abandoned, which is a continuation of U.S. Ser. No. 08/221,655, filedApr. 1, 1994, now abandoned, which is a continuation of U.S. Ser. No.07/967,622, filed Oct. 28, 1992, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/726,812, filed Jul. 8, 1991,now abandoned; a continuation-in-part of U.S. Ser. No. 08/376,062, filedJan. 20, 1995, now abandoned, which is a continuation of U.S. Ser. No.08/010,829, filed Jan. 29, 1993, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/726,812, filed Jul. 8, 1991,now abandoned; a continuation-in-part of U.S. Ser. No. 08/149,508, filedNov. 9, 1993, no abandoned, which is a continuation-in-part of U.S. Ser.No. 07/726,812, filed Jul. 8, 1991, now abandoned; acontinuation-in-part of U.S. Ser. No. 08/311,099, filed Sep. 23, 1994,no abandoned, which is a continuation-in-part of U.S. Ser. No.07/726,812, filed Jul. 8, 1991, now abandoned; and acontinuation-in-part of U.S. Ser. No. 08/338,730, filed Nov. 14, 1994,now abandoned, which is a continuation-in-part of U.S. Ser. No.07/726,812, filed Jul. 8, 1991, now abandoned.

FIELD OF THE INVENTION

This invention relates to a method for the in vitro culture andproliferation of multipotent neural stem cells, and to the use of thesecells and their progeny as tissue grafts. In one aspect, this inventionrelates to a method for the isolation and in vitro perpetuation of largenumbers of non-tumorigenic neural stem cell progeny which can be inducedto differentiate and which can be used for neurotransplantation in theundifferentiated or differentiated state, into an animal to alleviatethe symptoms of neurologic disease, neurodegeneration and centralnervous system (CNS) trauma. In another aspect, this invention relatesto a method of generating neural cells for the purposes of drugscreening of putative therapeutic agents targeted at the nervous system.In another aspect, this invention also relates to a method of generatingcells for autologous transplantation. In another aspect, the inventionrelates to a method for the in vivo proliferation and differentiation ofthe neural stem cell progeny in the host.

BACKGROUND OF THE INVENTION

The development of the mammalian central nervous system (CNS) begins inthe early stage of fetal development and continues until the post-natalperiod. The mature mammalian CNS is composed of neuronal cells(neurons), and glial cells (astrocytes and oligodendrocytes).

The first step in neural development is cell birth, which is the precisetemporal and spatial sequence in which stem cells and stem cell progeny(i.e daughter stem cells and progenitor cells) proliferate.Proliferating cells will give rise to neuroblasts, glioblasts and newstem cells.

The second step is a period of cell type differentiation and migrationwhen undifferentiated progenitor cells differentiate into neuroblastsand gliolblasts which give rise to neurons and glial cells which migrateto their final positions. Cells which are derived from the neural tubegive rise to neurons and glia of the CNS, while cells derived from theneural crest give rise to the cells of the peripheral nervous system(PNS). Certain factors present during development, such as nerve growthfactor (NGF), promote the growth of neural cells. NGF is secreted bycells of the neural crest and stimulates the sprouting and growth of theneuronal axons.

The third step in development occurs when cells acquire specificphenotypic qualities, such as the expression of particularneurotransmitters. At this time, neurons also extend processes whichsynapse on their targets. Neurons are generated primarily during thefetal period, while oligodendrocytes and astrocytes are generated duringthe early post-natal period. By the late post-natal period, the CNS hasits full complement of nerve cells.

The final step of CNS development is selective cell death, wherein thedegeneration and death of specific cells, fibers and synapticconnections "fine-tune" the complex circuitry of the nervous system.This "fine-tuning" continues throughout the life of the host. Later inlife, selective degeneration due to aging, infection and other unknownetiologies can lead to neurodegenerative diseases.

Unlike many other cells found in different tissues, the differentiatedcells of the adult mammalian CNS have little or no ability to enter themitotic cycle and generate new nerve cells. While it is believed thatthere is a limited and slow turnover of astrocytes (Korr et al., J.Comp. Neurol., 150:169, 1971) and that progenitors for oligodendrocytes(Wolsqijk and Noble, Development, 105:386, 1989) are present, thegeneration of new neurons does not normally occur. Neurogenesis, thegeneration of new neurons, is complete early in the postnatal period.However, the synaptic connections involved in neural circuits arecontinuously altered throughout the life of the individual, due tosynaptic plasticity and cell death. A few mammalian species (e.g. rats)exhibit the limited ability to generate new neurons in restricted adultbrain regions such as the dentate gyrus and olfactory bulb (Kaplan, J.Comp. Neurol., 195:323, 1981; Bayer, N.Y. Acad. Sci., 457:163, 1985).However, this does not apply to all mammals; and the generation of newCNS cells in adult primates does not occur (Rakic, Science, 227:1054,1985). This inability to produce new nerve cells in most mammals (andespecially primates) may be advantageous for long-term memory retention;however, it is a distinct disadvantage when the need to replace lostneuronal cells arises due to injury or disease.

The low turnover of cells in the mammalian CNS together with theinability of the adult mammalian CNS to generate new neuronal cells inresponse to the loss of cells following injury or disease has led to theassumption that the adult mammalian CNS does not contain multipotentneural stem cells.

The critical identifying feature of a stem cell is its ability toexhibit self-renewal or to generate more of itself. The simplestdefinition of a stem cell would be a cell with the capacity forself-maintenance. A more stringent (but still simplistic) definition ofa stem cell is provided by Potten and Loeffler (Development, 110:1001,1990) who have defined stem cells as "undifferentiated cells capable ofa) proliferation, b) self-maintenance, c) the production of a largenumber of differentiated functional progeny, d) regenerating the tissueafter injury, and e) a flexibility in the use of these options."

The role of stem cells is to replace cells that are lost by natural celldeath, injury or disease. The presence of stem cells in a particulartype of tissue usually correlates with tissues that have a high turnoverof cells. However, this correlation may not always hold as stem cellsare thought to be present in tissues (e.g., liver [Travis, Science,259:1829, 1993]) that do not have a high turnover of cells.

CNS disorders encompass numerous afflictions such as neurodegenerativediseases (e.g. Alzheimer's and Parkinson's), acute brain injury (e.g.stroke, head injury, cerebral palsy) and a large number of CNSdysfunctions (e.g. depression, epilepsy, and schizophrenia). In recentyears neurodegenerative disease has become an important concern due tothe expanding elderly population which is at greatest risk for thesedisorders. These diseases, which include Alzheimer's Disease, MultipleSclerosis (MS), Huntington's Disease, Amyotrophic Lateral Sclerosis, andParkinson's Disease, have been linked to the degeneration of neuralcells in particular locations of the CNS, leading to the inability ofthese cells or the brain region to carry out their intended function.

In addition to neurodegenerative diseases, acute brain injuries oftenresult in the loss of neural cells, the inappropriate functioning of theaffected brain region, and subsequent behavior abnormalities. Probablythe largest area of CNS dysfunction (with respect to the number ofaffected people) is not characterized by a loss of neural cells butrather by an abnormal functioning of existing neural cells. This may bedue to inappropriate firing of neurons, or the abnormal synthesis,release, and processing of neurotransmitters. These dysfunctions may bethe result of well studied and characterized disorders such asdepression and epilepsy, or less understood disorders such as neurosisand psychosis.

Degeneration in a brain region known as the basal ganglia can lead todiseases with various cognitive and motor symptoms, depending on theexact location. The basal ganglia consists of many separate regions,including the striatum (which consists of the caudate and putamen), theglobus pallidus, the substantia nigra, substantia innominate, ventralpallidum, nucleus basalis of Meynert, ventral tegmental area and thesubthalamic nucleus.

In the case of Alzheimer's Disease, there is a profound cellulardegeneration of the forebrain and cerebral cortex. In addition, uponcloser inspection, a localized degeneration in an area of the basalganglia, the nucleus basalis of Meynert, appears to be selectivelydegenerated. This nucleus normally sends cholinergic projections to thecerebral cortex which are thought to participate in cognitive functionsincluding memory.

Many motor deficits are a result of degeneration in the basal ganglia.Huntington's Chorea is associated with the degeneration of neurons inthe striatum, which leads to involuntary jerking movements in the host.Degeneration of a small region called the subthalamic nucleus isassociated with violent flinging movements of the extremities in acondition called ballismus, while degeneration in the putamen and globuspallidus is associated with a condition of slow writhing movements orathetosis. In the case of Parkinson's Disease, degeneration is seen inanother area of the basal ganglia, the substantia nigra par compacta.This area normally sends dopaminergic connections to the dorsal striatumwhich are important in regulating movement. Therapy for Parkinson'sDisease has centered upon restoring dopaminergic activity to thiscircuit.

Other forms of neurological impairment can occur as a result of neuraldegeneration, such as amyotrophic lateral sclerosis and cerebral palsy,or as a result of CNS trauma, such as stroke and epilepsy.

Demyelination of central and peripheral neurons occurs in a number ofpathologies and leads to improper signal conduction within the nervoussystems. Myelin is a cellular sheath, formed by glial cells, thatsurrounds axons and axonal processes that enhances variouselectrochemical properties and provides trophic support to the neuron.Myelin is formed by Schwann cells in the PNS and by oligodendrocytes inthe CNS. Among the various demyelinating diseases MS is the mostnotable.

To date, treatment for CNS disorders has been primarily via theadministration of pharmaceutical compounds. Unfortunately, this type oftreatment has been fraught with many complications including the limitedability to transport drugs across the blood-brain barrier and thedrug-tolerance which is acquired by patients to whom these drugs areadministered long-term. For instance, partial restoration ofdopaminergic activity in Parkinson's patients has been achieved withlevodopa, which is a dopamine precursor able to cross the blood-brainbarrier. However, patients become tolerant to the effects of levodopa,and therefore, steadily increasing dosages are needed to maintain itseffects. In addition, there are a number of side effects associated withlevodopa such as increased and uncontrollable movement.

Recently, the concept of neurological tissue grafting has been appliedto the treatment of neurological diseases such as Parkinson's Disease.Neural grafts may avert the need not only for constant drugadministration, but also for complicated drug delivery systems whicharise due to the blood-brain barrier. However, there are limitations tothis technique as well. First, cells used for transplantation whichcarry cell surface molecules of a differentiated cell from another hostcan induce an immune reaction in the host. In addition, the cells mustbe at a stage of development where they are able to form normal neuralconnections with neighboring cells. For these reasons, initial studieson neurotransplantation centered on the use of fetal cells. Perlow, etal. describe the transplantation of fetal dopaminergic neurons intoadult rats with chemically induced nigrostriatal lesions in "Braingrafts reduce motor abnormalities produced by destruction ofnigrostriatal dopamine system," Science 204:643-647 (1979). These graftsshowed good survival, axonal outgrowth and significantly reduced themotor abnormalities in the host animals.

In both human demyelinating diseases and rodent models there issubstantial evidence that demyelinated neurons are capable ofremyelination in vivo. In MS, for example, it appears that there areoften cycles of de- and remyelination. Similar observations in rodentdemyelinating paradigms lead to the prediction that exogenously appliedcells would be capable of remyelinating demyelinated axons.

This approach has proven successful in a number of experimentalconditions [Freidman et al., Brain Research, 378:142-146 (1986); Raine,et al., Laboratory Investigation 59:467-476 (1988); Duncan et al., J. ofNeurocytology, 17:351-360 (1988)]. The sources of cells for some ofthese experiments included dissociated glial cell suspensions preparedfrom spinal cords (Duncan et al., supra), Schwann cell cultures preparedfrom sciatic nerve [Bunge et al., 1992, WO 92/03536; Blakemore andCrang, J. Neurol. Sci., 70:207-223 (1985)]; cultures from dissociatedbrain tissue [Blakemore and Crang, Dev. Neurosci. 10: 1-11 (1988)],oligodendrocyte precursor cells [Gumpel et al., Dev. Neurosci.11:132-139 (1989)], O-2A cells [Wolswijk et al., Development 109:691-608(1990); Raff et al., Nature 3030:390-396 (1983); Hardy et al.,Development 111:1061-1080 (1991)], and immortalized O-2A cell lines,[Almazan and McKay Brain Res. 5 579:234-245 (1992)].

O-2A cells are glial progenitor cells which give rise in vitro only tooligodendrocytes and type II astrocytes. Cells which appear byimmunostaining in vivo to have the O-2A phenotype have been shown tosuccessfully remyelinate demyelinated neurons in vivo, [Godfraind etal., J. Cell Biol. 109:2405-2416 (1989)]. Injection of a large number ofO-2A cells is required to adequately remyelinate all targeted neurons invivo, since it appears that O-2A cells (like other glial cellpreparations) do not continue to divide in vivo. Although O-2Aprogenitor cells can be grown in culture, currently the only availableisolation technique employs optic nerve as starting material. This is alow yield source, which requires a number of purification steps. Thereis an additional drawback that O-2A cells isolated by the availableprocedures are capable of only a limited number of divisions [RaffScience 243:1450-1455 (1989)].

Although adult CNS neurons are not good candidates forneurotransplantation, neurons from the adult PNS have been shown tosurvive transplantation, and to exert neurotrophic and gliotrophiceffects on developing host neural tissue. One source of non-CNS neuraltissue for transplantation is the adrenal medulla. Adrenal chromaffincells originate from the neural crest like PNS neurons, and receivesynapses and produce carrier and enzyme proteins similar to PNS neurons.Although these cells function in an endocrine manner in the intactadrenal medulla, in culture these cells lose their glandular phenotypeand develop certain neural features in culture in the presence ofcertain growth factors and hormones [Notter, et al., "Neuronalproperties of monkey adrenal medulla in vitro, Cell Tissue Research244:69-76 (1986)]. When grafted into mammalian CNS, these cells surviveand synthesize significant quantities of dopamine which can interactwith dopamine receptors in neighboring areas of the CNS.

In U.S. Pat. No. 4,980,174, transplantation of monoamine-containingcells isolated from adult rat pineal gland and adrenal medulla into ratfrontal cortex led to the alleviation of learned helplessness, a form ofdepression in the host. In U.S. Pat. No. 4,753,635, chromaffin cells andadrenal medullary tissue derived from steers were implanted into thebrain stem or spinal cord of rats and produced analgesia when theimplanted tissue or cell was induced to release nociceptor interactingsubstances (i.e. catecholamines such as dopamine). Adrenal medullarycells have been autologously grafted into humans, and have survived,leading to mild to moderate improvement in symptoms (Watts, et al.,"Adrenal-caudate transplantation in patients with Parkinson's Disease(PD):1-year follow-up," Neurology 39 Suppl 1: 127 [1989], Hurtig, etal., "Postmortem analysis of adrenal-medulla-to-caudate autograft in apatient with Parkinson's Disease," Annals of Neurology 25: 607-614[1989]). However, adrenal cells do not obtain a normal neural phenotype,and are therefore probably of limited use for transplants where synapticconnections must be formed.

Another source of tissue for neurotransplantation is from cell lines.Cell lines are immortalized cells which are derived either bytransformation of normal cells with an oncogene (Cepko, "Immortalizationof neural cells via retrovirus-mediated oncogene transduction," Ann.Rev. Neurosci. 12:47-65 [1989]) or by the culturing of cells withaltered growth characteristics in vitro (Ronnett, et al., "Humancortical neuronal cell line: Establishment from a patient withunilateral megalencephaly," Science 248:603-605 [1990]). Such cells canbe grown in culture in large quantities to be used for multipletransplantations. Some cell lines have been shown to differentiate uponchemical treatment to express a variety of neuronal properties such asneurite formation, excitable membranes and synthesis ofneurotransmitters and their receptors. Furthermore, upondifferentiation, these cells appear to be amitotic, and thereforenoncancerous. However, the potential for these cells to induce adverseimmune responses, the use of retroviruses to immortalize cells, thepotential for the reversion of these cells to an amitotic state, and thelack of response of these cells to normal growth-inhibiting signals makecell lines less than optimal for widespread use.

Another approach to neurotransplantation involves the use of geneticallyengineered cell types or gene therapy. Using this method, a foreign geneor transgene can be introduced into a cell which is deficient in aparticular enzymatic activity, thereby allowing the cell to express thegene. Cells which now contain the transferred gene can be transplantedto the site of neurodegeneration, and provide products such asneurotransmitters and growth factors (Rosenberg, et al., "Graftinggenetically modified cells to the damaged brain: Restorative effects ofNGF Expression," Science 242:1575-1578, [1988]) which may function toalleviate some of the symptoms of degeneration. However, there stillexists a risk of inducing an immune reaction using currently availablecell lines. In addition, these cells may also not achieve normalneuronal connections with the host tissue.

Genetically modified cells have been used in neurological tissuegrafting in order to replace lost cells which normally produce aneurotransmitter. For example, fibroblasts have been geneticallymodified with a retroviral vector containing a cDNA for tyrosinehydroxylase, which allows them to produce dopamine, and implanted intoanimal models of Parkinson's Disease (Gage et al., U.S. Pat. No.5,082,670).

While the use of genetically modified fibroblasts to treat CNS disordershas shown promise in improving some behavioral deficits in animal modelsof Parkinson's Disease, and represents a novel approach to supplying aneeded transmitter to the CNS, it suffers from several significantdrawbacks as a treatment for Parkinson's Disease and in general as atherapeutic approach for treating neurodegenerative diseases and braininjury. First, the CNS is primarily composed of three cell typesneurons, astrocytes and oligodendrocytes. The implantation of a foreigncell such as a fibroblast into the CNS and its direct and indirecteffects on the functioning of the host cells has yet to be studied.However, it is likely that the expression of membrane bound factors andthe release of soluble molecules such as growth factors and proteaseswill alter the normal behavior of the surrounding tissue. This mayresult in the disruption of neuronal firing patterns either by a directaction on neurons or by an alteration in the normal functioning of glialcells.

Another concern that arises when fibroblasts are implanted into the CNSis the possibility that the implanted cells may lead to tumor formationbecause the intrinsic inhibition of fibroblast division is poorlycontrolled. Instead, extrinsic signals play a major role in controllingthe number of divisions the cell will undergo. The effect of the CNSenvironment on the division of implanted fibroblasts and the highprobability of a fibroblastic tumor formation has not been studied inthe long-term.

A third concern in transplanting fibroblasts into the CNS is thatfibroblasts are unable to integrate with the CNS cells as astrocytes,oligodendrocytes, or neurons do. Fibroblasts are intrinsically limitedin their ability to extend neuronal-like processes and form synapseswith host tissue. Hence, although the genetic modification andimplantation of fibroblasts into the CNS represents an improvement overthe current technology for the delivery of certain molecules to the CNS,the inability of fibroblasts to integrate and function as CNS tissue,their potential negative effects on CNS cells, and their limitedintrinsic control of proliferation limits their practical usage forimplantation for the treatment of acute or chronic CNS injury ordisease.

A preferred tissue for genetic modification and implantation would beCNS cells--neurons, astrocytes, or oligodendrocytes. One source of CNScells is from human fetal tissue. Several studies have shownimprovements in patients with Parkinson's Disease after receivingimplants of fetal CNS tissue. Implants of embryonic mesencephalic tissuecontaining dopamine cells into the caudate and putamen of human patientswas shown by Freed et al. (N Engl J Med 327:1549-1555 (1992)) to offerlong-term clinical benefit to some patients with advanced Parkinson'sDisease. Similar success was shown by Spencer et al. (N Engl J Med327:1541-1548 (1992)). Widner et al. (N Engl J Med 327:1556-1563 (1992))have shown long-term functional improvements in patients withMPTP-induced Parkinsonism that received bilateral implantation of fetalmesencephalic tissue.

While the studies noted above are encouraging, the use of largequantities of aborted fetal tissue for the treatment of disease raisesethical considerations and political obstacles. There are otherconsiderations as well. Fetal CNS tissue is composed of more than onecell type, and thus is not a well-defined source of tissue. In addition,there are serious doubts as to whether an adequate and constant supplyof fetal tissue would be available for transplantation. For example, inthe treatment of MPTP-induced Parkinsonism (Widner supra) tissue from 6to 8 fetuses were used for implantation into the brain of a singlepatient. There is also the added problem of the potential forcontamination during fetal tissue preparation. Moreover, the tissue mayalready be infected with a bacteria or virus, thus requiring expensivediagnostic testing for each fetus used. However, even diagnostic testingmight not uncover all infected tissue. For example, the diagnosis ofHIV-free tissue is not guaranteed because antibodies to the virus aregenerally not present until several weeks after infection.

While currently available transplantation approaches represent asignificant improvement over other available treatments for neurologicaldisorders, they suffer from significant drawbacks. The inability in theprior art of the transplant to fully integrate into the host tissue, andthe lack of availability of cells in unlimited amounts from a reliablesource for grafting are, perhaps, the greatest limitations ofneurotransplantation.

It would be more preferable to have a well-defined, reproducible sourceof neural tissue for transplantation that is available in unlimitedamounts. Since adult neural tissue undergoes minimal division, it doesnot readily meet these criteria. While astrocytes retain the ability todivide and are probably amenable to infection with foreign genes, theirability to form synapses with neuronal cells is limited and consequentlyso is their extrinsic regulation of the expression and release of theforeign gene product.

Oligodendrocytes suffer from some of the same problems. In addition,mature oligodendrocytes do not divide, limiting the infection ofoligodendrocytes to their progenitor cells (e.g. O2A cells). However,due to the limited proliferative ability of oligodendrocyte progenitors,the infection and harvesting of these cells does not represent apractical source.

The infection of neurons with foreign genes and implantation into theCNS would be ideal due to their ability to extend processes, makesynapses and be regulated by the environment. However, differentiatedneurons do not divide and transfection with foreign genes by chemicaland physical means is not efficient, nor are they stable for longperiods of time. The infection of primary neuronal precursors withretroviral vectors in vitro is not practical either because neuroblastsare intrinsically controlled to undergo a limited number of divisionsmaking the selection of a large number of neurons, that incorporate andexpress the foreign gene, nearly impossible. The possibility ofimmortalizing the neuronal precursors by retroviral transfer ofoncogenes and their subsequent infection of a desired gene is notpreferred due to the potential for tumor formation by the implantedcells.

In addition to the need for a well-defined, reproducible source ofneural cells available in unlimited amounts for transplantationpurposes, a similar need exists for drug screening purposes and for thestudy of CNS function, dysfunction, and development. The mature humannervous system is composed of billions of cells that are generatedduring development from a small number of precursors located in theneural tube. Due to the complexity of the mammalian CNS, the study ofCNS developmental pathways, as well as alterations that occur in adultmammalian CNS due to dysfunction, has been difficult. Such areas wouldbe better studied using relatively simple models of the CNS underdefined conditions.

Generally, two approaches have been taken for studying cultured CNScells: the use of primary neural cultures; and the use of neural celllines. Primary mammalian neural cultures can be generated from nearlyall brain regions providing that the starting material is obtained fromfetal or early post-natal animals. In general, three types of culturescan be produced, enriched either in neurons, astrocytes, oroligodendrocytes. Primary CNS cultures have proven valuable fordiscovering many mechanisms of neural function and are used for studyingthe effects of exogenous agents on developing and mature cells. Whileprimary CNS cultures have many advantages, they suffer from two primarydrawbacks. First, due to the limited proliferative ability of primaryneural cells, new cultures must be generated from several differentanimals. While great care is usually taken to obtain tissue at identicalstates of development and from identical brain regions, it is virtuallyimpossible to generate primary cultures that are identical. Hence, thereexists a significant degree of variability from culture to culture.

A second disadvantage of primary cultures is that the tissue must beobtained from fetuses or early post-natal animals. If primary culturesare to be performed on a regular basis, this requires the availabilityof a large source of starting material. While this is generally not aproblem for generating primary cultures from some species (e.g.rodents), it is for others (e.g. primates). Due to the limited supplyand ethical concerns, the culturing of primary cells from primates (bothhuman and non-human) is not practical.

Due to the limited proliferative ability of primary neural cells, thegeneration of a large number of homogenous cells for studies of neuralfunction, dysfunction, and drug design/screening has previously not beenachieved. Therefore, homogenous populations of cells that can generate alarge number of progeny for the in vitro investigation of CNS functionhas been studied by the use of cell lines. The generation of neural celllines can be divided into two categories: 1) spontaneously occurringtumors, and 2) custom-designed cell lines.

Of the spontaneously occurring tumors, probably the most studied cellline for neurobiology is the rat pheochromocytoma (PC12) cells that candifferentiate into sympathetic-like neurons in response to NGF. Thesecells have proven to be a useful model for studying mechanisms of neuraldevelopment and alterations (molecular and cellular) in response togrowth factors. Neuroblastoma and glioma cell lines have been used tostudy neuronal and glial functioning [Liles, et al., J. Neurosci. 7,2556-2563 (1987); Nister et al. Cancer Res. 48(14) 3910 (1988)].Embryonal carcinoma cells are derived from teratoma tumors of fetal germcells and have the ability to differentiate into a large number ofnon-neural cell types with some lines (e.g. P19 cells) [Jones-Villeneuveet al. J. Cell Biol. 94, 253-262 (1982)] having the ability todifferentiate into neural cells [(McBurney et al. J. Neurosci. 8(3)1063-73 (1993)]. A human teratocarcinoma-derived cell line, NTera2/cl.D1, with a phenotype resembling CNS neuronal precursor cells, canbe induced to differentiate in the presence of retinoic acid. However,the differentiated cells are restricted to a neuronal phenotype[Pleasure and Lee J. Neurosci. Res. 35: 585-602 (1993)]. While thesetypes of cell lines are able to generate a large number of cells forscreening the effects of exogenous agents on cell survival or function,the limited number of these types of lines, the limited number ofphenotypes that they are able to generate and the unknown nature oftheir immortalization (which may effect the function of the cells in anundefined manner) makes these types of cell lines less than ideal for invitro models of neural function and discovery of novel therapeutics.

An alternative approach to spontaneously occurring cell lines is theintentional immortalization of a primary cell by introducing an oncogenethat alters the genetic make-up of the cell thereby inducing the cell toproliferate indefinitely. This approach has been used by many groups togenerate a number of interesting neural cell lines [(Bartlett et al.Proc. Nat. Acad. Sci. 85(9) 3255-3259 (1988); Frederiksen et al. Neuron1, 439-448 (1988); Trotter et al. Oncogene 4: 457-464 (1989); Ryder etal. J. Neurobiol. 21: 356-375 (1980); Murphy et al. J. Neurobiol 22:522-535 (1991); Almazan and McKay et al. Brain Res. 579: 234-245(1992)]. While these lines may prove useful for studying the decisionsthat occur during cell determination and differentiation, and fortesting the effects of exogenous agents, they suffer from severaldrawbacks. First, the addition of an oncogene that alters theproliferative status of a cell may affect other properties of the cell(oncogenes may play other roles in cells besides regulating the cellcycle). This is well illustrated in a study by Almazan and McKay, supra,and their immortalization of an oligodendrocyte precursor from the opticnerve which is unable to differentiate into type II astrocytes(something that normal optic nerve oligodendrocyte precursors can do).The authors suggest the presence of the immortalizing antigen may alterthe cells ability to differentiate into astrocytes.

Another drawback to using intentionally immortalized cells results fromthe fact that the nervous system is composed of billions of cells andpossibly thousands of different cell types, each with unique patterns ofgene expression and responsiveness to their environment. Acustom-designed cell line is the result of the immortalization of asingle progenitor cell and its clonal expansion. While a large supply ofone neural cell type can be generated, this approach does not take intoaccount cellular interactions between different cell types. In addition,while it is possible to immortalize cells from a given brain region,immortalization of a desired cell is not possible due to the lack ofcontrol over which cells will be altered by the oncogene. Hence, whilecustom designed cell lines offer a few advantages over spontaneouslyoccurring tumors, they suffer from several drawbacks and are less thanideal for understanding CNS function and dysfunction.

Therefore, in view of the aforementioned deficiencies attendant withprior art methods of neural cell culturing, transplantation, and CNSmodels, a need exists in the art for a reliable source of unlimitednumbers of undifferentiated neural cells for neurotransplantation anddrug screening which are capable of differentiating into neurons,astrocytes, and oligodendrocytes. Preferably cellular division in suchcells from such a source would be epigenetically regulated and asuitable number of cells could be efficiently prepared in sufficientnumbers for transplantation. The cells should be suitable in autografts,xenografts, and allografts without a concern for tumor formation. Thereexists a need for the isolation, perpetuation and transplantation ofautologous neural cells from the juvenile or adult brain that arecapable of differentiating into neurons and glia.

A need also exists for neural cells, capable of differentiating intoneurons, astrocytes and oligodendrocytes that are capable ofproliferation in vitro and thus amenable to genetic modificationtechniques.

Additionally, there exists a need for the repair of damaged neuraltissue in a relatively non-invasive fashion, that is by inducing neuralcells to proliferate and differentiate into neurons, astrocytes, andoligodendrocytes in vivo, thereby averting the need for transplantation.

Accordingly, a major object of the present invention is to provide areliable source of an unlimited number of neural cells forneurotransplantation that are capable of differentiating into neurons,astrocytes, and oligodendrocytes.

It is another object of the present invention to provide a method forthe in vitro proliferation of neural stem cells from embryonic, juvenileand adult brain tissue, to produce unlimited numbers of precursor cellsavailable for transplantation that are capable of differentiating intoneurons, astrocytes, and oligodendrocytes.

A further object of the invention is to provide methods for inducingneural cells to proliferate and differentiate in vivo, thereby avertingthe need for neurotransplantation.

A still further object of the invention is to provide a method ofgenerating large numbers of normal neural cells for the purpose ofscreening putative therapeutic agents targeted at the nervous system andfor models of CNS development, function, and dysfunction.

SUMMARY OF THE INVENTION

This invention provides in one aspect a composition for inducing theproliferation of a multipotent neural stem cell comprising a culturemedium supplemented with at least one growth factor, preferablyepidermal growth factor or transforming growth factor alpha.

The invention also provides a method for the in vitro proliferation anddifferentiation of neural stem cells and stem cell progeny comprisingthe steps of (a) isolating the cell from a mammal, (b) exposing the cellto a culture medium containing a growth factor, (c) inducing the cell toproliferate, and (d) inducing the cell to differentiate. Proliferationand perpetuation of the neural stem cell progeny can be carried outeither in suspension cultures, or by allowing cells to adhere to a fixedsubstrate. Proliferation and differentiation can be done before or aftertransplantation, and in various combinations of in vitro or in vivoconditions, including (1) proliferation and differentiation in vitro,then transplantation, (2) proliferation in vitro. transplantation, thenfurther proliferation and differentiation in vivo, and (3) proliferationin vitro, transplantation and differentiation in vivo.

The invention also provides for the proliferation and differentiation ofthe progenitor cells in vivo, which can be done directly in the hostwithout the need for transplantation.

The invention also provides a method for the in vivo transplantation ofneural stem cell progeny, treated as in any of (1) through (3) above,which comprises implanting, into a mammal, these cells which have beentreated with at least one growth factor.

Furthermore, the invention provides a method for treatingneurodegenerative diseases comprising administering to a mammal neuralstem cell progeny which have been treated as in any of (1) through (3),and induced to differentiate into neurons and/or glia.

The invention also provides a method for treating neurodegenerativedisease comprising stimulating in vivo mammalian CNS neural stem cellsto proliferate and the neural stem cell progeny to differentiate intoneurons and/or glia.

The invention also provides a method for the transfection of neural stemcells and stem cell progeny with vectors which can express the geneproducts for growth factors, growth factor receptors, and peptideneurotransmitters, or express enzymes which are involved in thesynthesis of neurotransmitters, including those for amino acids,biogenic amines and neuropeptides, and for the transplantation of thesetransfected cells into regions of neurodegeneration.

In a still further aspect, the invention provides a method for thescreening of potential neurologically therapeutic pharmaceuticals usingneural stem cell progeny which have been proliferated in vitro.

BRIEF DESCRIPTION OF THE FIGS.

FIG. 1: Diagram Illustrating the Proliferation of a Multipotent NeuralStem Cell

(A) In the presence of a proliferation-inducing growth factor the stemcell divides and gives rise to a sphere of undifferentiated cellscomposed of more stem cells and progenitor cells. (B) When the clonallyderived sphere of undifferentiated cells is dissociated and plated assingle cells, on a non-adhesive substrate and in the presence of aproliferation-inducing growth factor, each stem cell will generate a newsphere. (C) If the spheres are cultured in conditions that allowdifferentiation, the progenitor cells differentiate into neurons,astrocytes and oligodendrocytes.

FIGS. 2A-2D: Proliferation Of Epidermal Growth Factor (EGF) ResponsiveCells

After 2 days in vitro EGF-responsive cells begin to proliferate (FIG.2A). After 4 days in vitro small clusters of cells known as neurospheresare apparent (FIG. 2B). The neurospheres of continuously proliferatingcells continue to grow in size (FIG. 2C) until they lift off thesubstrate and float in suspension (FIG. 2D). At this stage, the floatingspheres can be easily removed, dissociated into single cells and, in thepresence of EGF, proliferation can be re-initiated. (Bar: 50 μm).

FIGS. 3A-3D: Differentiation Of Cells From Single EGF-Generated SpheresInto Neurons, Astrocytes, And Oligodendrocytes

Triple-label immunocytochemistry with antibodies to microtubuleassociated protein (MAP-2), glial fibrillary acidic protein (GFAP), andO4 (a cell surface antigen) are used to detect the presence of neurons(FIG. 3B), astrocytes (FIG. 3C) and oligodendrocytes (FIG. 3D),respectively, from an EGF-generated, stem cell-derived neurosphere (FIG.3A) derived from primary culture. (Bar: 50 μm).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for inducing multipotent neuralstem cells from fetal, juvenile, or adult mammalian tissue toproliferate in vitro or in vivo (i.e. in situ), to generate largenumbers of neural stem cell progeny capable of differentiating intoneurons, astrocytes, and oligodendrocytes. Methods for differentiationof the neural stem cell progeny are also provided. The induction ofproliferation and differentiation of neural stem cells can be doneeither by culturing the cells in suspension or on a substrate onto whichthey can adhere. Alternatively, proliferation and differentiation ofneural stem cells can be induced, under appropriate conditions, in thehost in the following combinations: (1) proliferation anddifferentiation in vitro, then transplantation, (2) proliferation invitro, transplantation, then further proliferation and differentiationin vivo, (3) proliferation in vitro, transplantation and differentiationin vivo, and (4) proliferation and differentiation in vivo.Proliferation and differentiation in vivo (i.e. in situ) can involve anon-surgical approach that coaxes neural stem cells to proliferate invivo with pharmaceutical manipulation. Thus, the invention provides ameans for generating large numbers of undifferentiated anddifferentiated neural cells for neurotransplantation into a host inorder to treat neurodegenerative disease and neurological trauma, fornon-surgical methods of treating neurodegenerative disease andneurological trauma, and for drug-screening applications.

Multipotent Neural Stem Cells

Neurobiologists have used various terms interchangeably to describe theundifferentiated cells of the CNS. Terms such as "stem cell", "precursorcell" and "progenitor cell" are commonly used in the scientificliterature. However, there are different types of undifferentiatedneural cells, with differing characteristics and fates. U.S. Ser. No.08/270,412 abandoned, which is a continuation application of U.S. Ser.No. 07/726,812, abandoned, termed the cells obtained and proliferatedusing the methods of Examples 1-4 below "progenitor cells". Theterminology used for undifferentiated neural cells has evolved such thatthese cells are now termed "neural stem cells". U.S. Ser. No. 08/270,412abandoned defines the "progenitor" cell proliferated in vitro to mean"an oligopotent or multipotent stem cell which is able to divide withoutlimit and under specific conditions can produce daughter cells whichterminally differentiate into neurons and glia." The capability of acell to divide without limit and produce daughter cells which terminallydifferentiate into neurons and glia are stem cell characteristics.Accordingly, as used herein, the cells proliferated using the methodsdescribed in Examples 1-4 are termed "neural stem cells". A neural stemcell is an undifferentiated neural cell that can be induced toproliferate using the methods of the present invention. The neural stemcell is capable of self-maintenance, meaning that with each celldivision, one daughter cell will also be a stem cell. The non-stem cellprogeny of a neural stem cell are termed progenitor cells. Theprogenitor cells generated from a single multipotent neural stem cellare capable of differentiating into neurons, astrocytes (type I and typeII) and oligodendrocytes. Hence, the neural stem cell is "multipotent"because its progeny have multiple differentiative pathways.

The term "neural progenitor cell", as used herein, refers to anundifferentiated cell derived from a neural stem cell, and is not itselfa stem cell. Some progenitor cells can produce progeny that are capableof differentiating into more than one cell type. For example, an O-2Acell is a glial progenitor cell that gives rise to oligodendrocytes andtype II astrocytes, and thus could be termed a "bipotential" progenitorcell. A distinguishing feature of a progenitor cell is that, unlike astem cell, it has limited proliferative ability and thus does notexhibit self-maintenance. It is committed to a particular path ofdifferentiation and will, under appropriate conditions, eventuallydifferentiate into glia or neurons.

The term "precursor cells", as used herein, refers to the progeny ofneural stem cells, and thus includes both progenitor cells and daughterneural stem cells.

Neural stem cell progeny can be used for transplantation into aheterologous, autologous, or xenogeneic host. Multipotent neural stemcells can be obtained from embryonic, post-natal, juvenile or adultneural tissue. The neural tissue can be obtained from any animal thathas neural tissue such as insects, fish, reptiles, birds, amphibians,mammals and the like. The preferred source neural tissue is frommammals, preferably rodents and primates, and most preferably, mice andhumans.

In the case of a heterologous donor animal, the animal may beeuthanized, and the neural tissue and specific area of interest removedusing a sterile procedure. Areas of particular interest include any areafrom which neural stem cells can be obtained that will serve to restorefunction to a degenerated area of the host's nervous system,particularly the host's CNS. Suitable areas include the cerebral cortex,cerebellum, midbrain, brainstem, spinal cord and ventricular tissue, andareas of the PNS including the carotid body and the adrenal medulla.Preferred areas include regions in the basal ganglia, preferably thestriatum which consists of the caudate and putamen, or various cellgroups such as the globus pallidus, the subthalamic nucleus, the nucleusbasalis which is found to be degenerated in Alzheimer's Diseasepatients, or the substantia nigra pars compacta which is found to bedegenerated in Parkinson's Disease patients. Particularly preferredneural tissue is obtained from ventricular tissue that is found liningCNS ventricles and includes the subependyma. The term "ventricle" refersto any cavity or passageway within the CNS through which cerebral spinalfluid flows. Thus, the term not only encompasses the lateral, third, andfourth ventricles, but also encompasses the central canal, cerebralaqueduct, and other CNS cavities. Human heterologous neural stem cellsmay be derived from fetal tissue following elective abortion, or from apost-natal, juvenile or adult organ donor. Autologous neural tissue canbe obtained by biopsy, or from patients undergoing neurosurgery in whichneural tissue is removed, for example, during epilepsy surgery, temporallobectomies and hippocampalectomies. Neural stem cells have beenisolated from a variety of adult CNS ventricular regions, including thefrontal lobe, conus medullaris, thoracic spinal cord, brain stem, andhypothalamus, and proliferated in vitro using the methods detailedherein. In each of these cases, the neural stem cell exhibitsself-maintenance and generates a large number of progeny which includeneurons, astrocytes and oligodendrocytes.

Normally, the adult mammalian CNS is mitotically quiescent in vivo withthe exception of the subependymal region lining the lateral ventriclesin the forebrain.

This region contains a subpopulation of constitutively proliferatingcells with a cell cycle time of 12.7 hours. BrdU and retroviral labelingof the proliferating cells reveal that none of the newly generated cellsdifferentiate into mature neurons or glia nor do they migrate into otherCNS regions (Morshead and Van der Kooy, supra).

The continual proliferation and maintenance of a constant number ofcells within the subependyma is explained by two mechanisms. The deathof one of the daughter cells after each division maintains theproliferating population at a constant number. The constitutivelydividing population eventually dies out (and hence is not a stem cellpopulation) however, a subpopulation of relatively quiescent cellswithin the subependyma is able to repopulate the constitutively dividingpopulation. This stem cell-like mode of maintaining the proliferativesubependymal population is analogous to other tissues where cells have ashort life span and are repopulated by a subpopulation of relativelyquiescent cells referred to as stem cells.

As detailed in Example 27, experiments utilizing retrovirus infection ofconstituitively proliferating cells in vivo and subsequentβ-galactosidase (β-gal) reporter gene expression as a non-dilutingmarker show that with increasing adult mice survival times (of up to 28days post retrovirus infection) there is a progressive loss of β-galpositive subependymal cells. Relative to 1 day survival animals, 6 daysfollowing retrovirus injection there is a 45% loss of β-gal positivecells and 28 days following retrovirus infection there is a 97% loss.Using nested polymerase chain reaction (PCR) to identify single cellscontaining retroviral DNA it was determined that the loss of B-galexpressing cells is due to the loss of the retrovirally infected cellsthrough cell death, not due to the turn-off of β-gal expression.

Intraperitoneal injections of BrdU (a thymidine analog that isincorporated into the DNA of dividing cells) reveal that 33% of thecells within some regions of the subependyma make up the normallyconstituitively dividing population (see Morshead and van der Kooy, J.Neurosci. 12:249 (1992)). The number of BrdU labelled cells decreasesover time. By 30 days after BrdU labeling, only 3% of the dividing cellsare still labelled. The heavy labeling of only a small number of cells30 days after BrdU injections demonstrates that although the labelledcells were dividing at the time of the injections they were relativelyquiescent for the 30 day period. This suggests that these few labeledcells are stem cells rather than cells of the constitutivelyproliferating population.

The above two examples support the hypothesis that the maintenance ofthe constant number of proliferating subependymal cells seen throughoutadult life requires the presence of a relatively quiescent stem cellthat proliferates sporadically to replenish the constitutivelyproliferating population and to self-renew.

As detailed in Example 24, the constitutively dividing subependymalcells can be killed off by injecting high doses of radioactive thymidinefor the duration of the cell cycle at intervals less than S-phaseduration. At one day post-kill the proliferating population is 10% ofcontrols and by 8 days the proliferating population is back to controllevels. If the replenished population is due to the recruitment ofnormally quiescent stem cells into the proliferative mode, then a secondkill at the time that stem cells are generating progeny to repopulatethe subependyma should alter the number of cells within theconstitutively proliferating population. When a second kill is done 2days after the initial kill, 8 days later the constitutivelyproliferating population is only 45% of the control values (animalsreceiving no thymidine kill treatment) or animals that received only onekill at day 0 (the time of the first kill). The reduction in the numberof proliferative cells in the subependyma is maintained at 63% even at31 days after the second kill. When a second kill is done on day 4, theproliferating population returns to 85% of control values 8 days later.These results suggest that the normally quiescent stem cell is recruitedinto the proliferative mode within the first two days after the initialkill and that by 4 days the stem cell no longer needs to be recruited torepopulate the subependyma.

As detailed in Example 26 below, an experiment was performed todetermine whether the in vitro stem cell is derived from theconstitutively proliferating population or from the quiescentpopulation. Animals were treated in one of the following ways:

Group 1. Control

High doses of radioactive thymidine were given on:

Group 2. day 0

Group 3. day 0 and day 2

Group 4. day 0 and day 4

16 to 20 following the last injection animals were killed and stem cellsisolated from the striatum (including the subependymal region) via themethods described in Example 2 below.

In groups 2-4 the constitutively proliferating population was killed. Ingroup 3 stem cells that are recruited into the cell cycle to repopulatethe subependymal proliferating cells were also killed.

Number of Neurospheres produced in vitro:

Group 1. 100% (Control)

Group 2. 100%

Group 3. 45%

Group 4. 85%

These results demonstrate that when you eliminate nearly all of theconstitutively proliferating cells in the subependyma this does notaffect the number of stem cells that can be isolated and proliferated invitro (group 1 vs. group 2 and 4). However, when the normally quiescentcells are killed when they are recruited to repopulate the subependyma(as with group 3) the number of stem cells that can be isolated in vitrois significantly reduced (group 3 vs. group 1 and 2). By 4 days afterthe first kill most of the stem cells themselves are no longer turningover and as a result are not killed by the second series of tritiatedthymidine injections (hence, only a 15% reduction [group 4] compared to55% reduction [group 3]).

The above results demonstrate that, in adult, the stem cells which areproliferated in vitro are derived from the quiescent population ofsubependymal cells in vivo. This also explains why stem cells can bederived from CNS ventricular regions, other than the forebrain, which donot have a subpopulation of constitutively proliferating cells.

In Vitro Proliferation of Neural Stem Cells

Cells can be obtained from donor tissue by dissociation of individualcells from the connecting extracellular matrix of the tissue. Tissuefrom a particular neural region is removed from the brain using asterile procedure, and the cells are dissociated using any method knownin the art including treatment with enzymes such as trypsin, collagenaseand the like, or by using physical methods of dissociation such as witha blunt instrument. Dissociation of fetal cells can be carried out intissue culture medium, while a preferable medium for dissociation ofjuvenile and adult cells is low Ca²⁺ artificial cerebral spinal fluid(aCSF). Regular aCSF contains 124 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 2 mMCaCl₂, 26 mM NaHCO₃, and 10 mM D-glucose. Low Ca²⁺ aCSF contains thesame ingredients except for MgCl₂ at a concentration of 3.2 mM and CaCl₂at a concentration of 0.1 mM. Dissociated cells are centrifuged at lowspeed, between 200 and 2000 rpm, usually between 400 and 800 rpm, andthen resuspended in culture medium. The neural cells can be cultured insuspension or on a fixed substrate. However, substrates tend to inducedifferentiation of the neural stem cell progeny. Thus, suspensioncultures are preferred if large numbers of undifferentiated neural stemcell progeny are desired. Cell suspensions are seeded in any receptaclecapable of sustaining cells, particularly culture flasks, culture platesor roller bottles, and more particularly in small culture flasks such as25 cm² culture flasks. Cells cultured in suspension are resuspended atapproximately 5×10⁴ to 2×10⁵ cells/ml, preferably 1×10 cells/ml. Cellsplated on a fixed substrate are plated at approximately 2-3×10³cells/cm², preferably 2.5×10³ cells/cm.

The dissociated neural cells can be placed into any known culture mediumcapable of supporting cell growth, including HEM, DMEM, RPMI, F-12, andthe like, containing supplements which are required for cellularmetabolism such as glutamine and other amino acids, vitamins, mineralsand useful proteins such as transferrin and the like. Medium may alsocontain antibiotics to prevent contamination with yeast, bacteria andfungi such as penicillin, streptomycin, gentamicin and the like. In somecases, the medium may contain serum derived from bovine, equine, chickenand the like. However, a preferred embodiment for proliferation ofneural stem cells is to use a defined, serum-free culture medium, asserum tends to induce differentiation and contains unknown components(i.e. is undefined). A defined culture medium is also preferred if thecells are to be used for transplantation purposes. A particularlypreferable culture medium is a defined culture medium comprising amixture of DMEM, F12, and a defined hormone and salt mixture. Thisculture medium is referred to herein as "Complete Medium" and isdescribed in detail in Example 3.

Conditions for culturing should be close to physiological conditions.The pH of the culture medium should be close to physiological pH,preferably between pH 68, more preferably between about pH 7 to 7.8,with pH 7.4 being most preferred. Physiological temperatures rangebetween about 30° C. to 40° C. Cells are preferably cultured attemperatures between about 32° C. to about 38° C., and more preferablybetween about 35° C. to about 37° C.

The culture medium is supplemented with at least oneproliferation-inducing growth factor. As used herein, the term "growthfactor" refers to a protein, peptide or other molecule having a growth,proliferative, differentiative, or trophic effect on neural stem cellsand/or neural stem cell progeny. Growth factors which may be used forinducing proliferation include any trophic factor that allows neuralstem cells and precursor cells to proliferate, including any moleculewhich binds to a receptor on the surface of the cell to exert a trophic,or growth-inducing effect on the cell. Preferred proliferation-inducinggrowth factors include EGF, amphiregulin, acidic fibroblast growthfactor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2),transforming growth factor alpha (TGFα), and combinations thereof.

Preferred proliferation-inducing growth factors include EGF and TGFα. Apreferred combination of proliferation-inducing growth factors is EGF orTGFα with FGF-1 or FGF-2. Growth factors are usually added to theculture medium at concentrations ranging between about 1 fg/ml to 1mg/ml. Concentrations between about 1 to 100 ng/ml are usuallysufficient. Simple titration experiments can be easily performed todetermine the optimal concentration of a particular growth factor.

In addition to proliferation-inducing growth factors, other growthfactors may be added to the culture medium that influence proliferationand differentiation of the cells including NGF, platelet-derived growthfactor (PDGF), thyrotropin releasing hormone (TRH), transforming growthfactor betas (TGFβs), insulin-like growth factor (IGF₋₁) and the like.

Within 3-4 days in the presence of a proliferation-inducing growthfactor, a multipotent neural stem cell begins to divide giving rise to acluster of undifferentiated cells referred to herein as a "neurosphere".The cells of a single neurosphere are clonal in nature because they arethe progeny of a single neural stem cell. In the continued presence of aproliferation-inducing growth factor such as EGF or the like, precursorcells within the neurosphere continue to divide resulting in an increasein the size of the neurosphere and the number of undifferentiated cells.The neurosphere is not immunoreactive for GFAP, neurofilament (NF),neuron-specific enolase (NSE) or myelin basic protein (MBP). However,precursor cells within the neurosphere are immunoreactive for nestin, anintermediate filament protein found in many types of undifferentiatedCNS cells. The nestin marker was characterized by Lehndahl et al., Cell60:585-595 (1990). Antibodies are available to identify nestin,including the rat antibody referred to as Rat401. The mature phenotypesassociated with the differentiated cell types that may be derived fromthe neural stem cell progeny are predominantly negative for the nestinphenotype.

After about 4 to 5 days in the absence of a substrate, the proliferatingneurospheres lift off the floor of the culture dish and tend to form thefree-floating clusters characteristic of neurospheres. Floatingneurospheres are depicted in FIG. 2d. It is possible to vary the cultureconditions so that while the precursor cells still express the nestinphenotype, they do not form the characteristic neurospheres. Theproliferating precursor cells of the neurosphere continue to proliferatein suspension. After about 3-10 days in vitro, and more particularlyafter about 6-7 days in vitro, the proliferating neurospheres are fedevery 2-7 days, preferably every 2-4 days by gentle centrifugation andresuspension in Complete Medium containing a growth factor.

The neurospheres of the suspension culture can be easily passaged toreinitiate proliferation. After 6-7 days in vitro, the culture flasksare shaken well and the neurospheres allowed to settle on the bottomcorner of the flask. The neurospheres are then transferred to a 50 mlcentrifuge tube and centrifuged at low speed. The medium is aspirated,and the neurospheres are resuspended in a small amount of CompleteMedium. Individual cells in the neurospheres can be separated byphysical dissociation of the neurospheres with a blunt instrument, forexample, by triturating the neurospheres with a pipette, especially afire polished pasteur pipette, to form a single cell suspension ofneural stem cell progeny. The cells are then counted and replated at thedesired density to reinitiate proliferation. Single cells from thedissociated neurospheres are suspended in Complete Medium containinggrowth factor, and a percentage of these cells proliferate and form newneurospheres largely composed of undifferentiated cells. This procedurecan be repeated weekly to result in a logarithmic increase in the numberof viable cells at each passage. The procedure is continued until thedesired number of precursor cells is obtained.

The number of neural stem cell progeny proliferated in vitro from themammalian CNS can be increased dramatically by injecting a growth factoror combination of growth factors, for example EGF, FGF, or EGF and FGFtogether, into the ventricles of the donor in vivo using the in vivoproliferation methods described in more detail below. As detailed inExample 31 below, 6 days after infusion of EGF into the lateralventricle of a mouse forebrain, the walls of the ventricle were removedand the stem cells harvested. Infusion of EGF into the lateral ventricleincreased the efficiency of the yield of stem cells that proliferated toform neurospheres.

This ability to enhance the proliferation of neural stem cells shouldprove invaluable when stem cells are to be harvested for latertransplantation back into a patient, thereby making the initialsurgery 1) less traumatic because less tissue would have to be removedand 2) more efficient because a greater yield of stem cells per surgerywould proliferate in vitro.

Additionally, the patient's stem cells, once they have proliferated invitro, could also be genetically modified in vitro using the techniquesdescribed below. The in vitro genetic modification may be more desirablein certain circumstances than in vivo genetic modification techniqueswhen more control over the infection with the genetic material isrequired.

Neural stem cell progeny can be cryopreserved until they are needed byany method known in the art. The cells can be suspended in an isotonicsolution, preferably a cell culture medium, containing a particularcryopreservant. Such cryopreservants include dimethyl sulfoxide (DMSO),glycerol and the like. These cryopreservants are used at a concentrationof 5-15%, preferably 8-10%. Cells are frozen gradually to a temperatureof -10° C. to, preferably -20° C. to -100° C., and more preferably -70°C. to -80° C.

Differentiation of Neural Stem Cell Progeny

Differentiation of the cells can be induced by any method known in theart which activates the cascade of biological events which lead togrowth, which include the liberation of inositol triphosphate andintracellular Ca²⁺, liberation of diacyl glycerol and the activation ofprotein kinase C and other cellular kinases, and the like. Treatmentwith phorbol esters, differentiation-inducing growth factors and otherchemical signals can induce differentiation. Differentiation can also beinduced by plating the cells on a fixed substrate such as flasks,plates, or coverslips coated with an ionically charged surface such aspoly-L-lysine and poly-L-ornithine and the like.

Other substrates may be used to induce differentiation such as collagen,fibronectin, laminin, MATRIGEL, and the like. Differentiation can alsobe induced by leaving the cells in suspension in the presence of aproliferation-inducing growth factor, without reinitiation ofproliferation (i.e. without dissociating the neurospheres).

A preferred method for inducing differentiation of the neural stem cellprogeny comprises culturing the cells on a fixed substrate in a culturemedium that is free of the proliferation-inducing growth factor. Afterremoval of the proliferation-inducing growth factor, the cells adhere tothe substrate (e.g. poly-ornithine-treated plastic or glass), flatten,and begin to differentiate into neurons and glial cells. At this stagethe culture medium may contain serum such as 0.5-1.0% fetal bovine serum(FBS). However, for certain uses, if defined conditions are required,serum would not be used. Within 2-3 days, most or all of the neural stemcell progeny begin to lose immunoreactivity for nestin and begin toexpress antigens specific for neurons, astrocytes or oligodendrocytes asdetermined by immunocytochemistry techniques well known in the art.

Immunocytochemistry (e.g. dual-label immunofluorescence andimmunoperoxidase methods) utilizes antibodies that detect cell proteinsto distinguish the cellular characteristics or phenotypic properties ofneurons from astrocytes and oligodendrocytes. In particular, cellularmarkers for neurons include NSE, NF, β-tub, MAP-2; and for glia, GFAP(an identifier of astrocytes), galactocerebroside (GalC) (a myelinglycolipid identifier of oligodendrocytes), and the like.

Immunocytochemistry can also be used to detect the expression ofneurotransmitters, or in some cases the expression of enzymesresponsible for neurotransmitter synthesis. For the identification ofneurons, antibodies can be used that detect the presence ofacetylcholine (ACh), dopamine, epinephrine, norepinephrine, histamine,serotonin or 5-hydroxytryptamine (5-HT), neuropeptides such as substanceP, adrenocorticotrophic hormone, vasopressin or anti-diuretic hormone,oxytocin, somatostatin, angiotensin II, neurotensin, and bombesin,hypothalamic releasing hormones such as TRH and luteinizing releasinghormone, gastrointestinal peptides such as vasoactive intestinal peptide(VIP) and cholecystokinin (CCK) and CCK-like peptide, opioid peptidessuch as endorphins like β-endorphin and enkephalins such as met- andleu-enkephalin, prostaglandins, amino acids such as y-amino butyric acid(GABA), glycine, glutamate, cysteine, taurine and aspartate anddipeptides such as carnosine. Antibodies toneurotransmitter-synthesizing enzymes can also be used such as glutamicacid decarboxylase (GAD) which is involved in the synthesis of GABA,choline acetyltransferase (ChAT) for ACh synthesis, dopa decarboxylase(DDC) for dopamine, dopamine-β-hydroxylase (DBH) for norepinephrine, andamino acid decarboxylase for 5-HT. Antibodies to enzymes that areinvolved in the deactivation of neurotransmitters may also be usefulsuch as acetyl cholinesterase (AChE) which deactivates ACh. Antibodiesto enzymes involved in the reuptake of neurotransmitters into neuronalterminals such as monoamine oxidase and catechol-o-methyl transferasefor dopamine, for 5-HT, and GABA transferase for GABA may also identifyneurons. Other markers for neurons include antibodies toneurotransmitter receptors such as the AChE nicotinic and muscarinicreceptors, adrenergic receptors α¹, α₂, β¹ and α₂, the dopamine receptorand the like. Cells that contain a high level of melanin, such as thosefound in the substantia nigra, could be identified using an antibody tomelanin.

In situ hybridization histochemistry can also be performed, using cDNAor RNA probes specific for the peptide neurotransmitter or theneurotransmitter synthesizing enzyme mRNAs. These techniques can becombined with immunocytochemical methods to enhance the identificationof specific phenotypes. If necessary, the antibodies and molecularprobes discussed above can be applied to Western and Northern blotprocedures respectively to aid in cell identification.

A preferred method for the identification of neurons usesimmunocytochemistry to detect immunoreactivity for NSE, NF, NeuN, andthe neuron specific protein, tau-1. Because these markers are highlyreliable, they will continue to be useful for the primary identificationof neurons, however neurons can also be identified based on theirspecific neurotransmitter phenotype as previously described.

Type I astrocytes, which are differentiated glial cells that have aflat, protoplasmic/fibroblast-like morphology, are preferably identifiedby their immunoreactivity for GFAP but not A2B5. Type II astrocytes,which are differentiated glial cells that display a stellateprocess-bearing morphology, are preferably identified usingimmunocytochemistry by their phenotype GFAP(+), A2B5(+) phenotype.

Cells that do not express intermediate filaments specific for neurons orfor astrocytes, begin to express markers specific for oligodendrocytesin a correct temporal fashion. That is, the cells first becomeimmunoreactive for O4, galactocerebroside (GalC, a myelin glycolipid)and finally, MBP. These cells also possess a characteristicoligodendrocyte morphology.

The present invention provides a method of influencing the relativeproportion of these differentiated cell types by the addition ofexogenous growth factors during the differentiation stage of theprecursor cells. By using dual-label immunofluorescence andimmunoperoxidase methods with various neuronal- and glial-specificantibodies, the effect of the exogenous growth factors on thedifferentiating cells can be determined.

The biological effects of growth and trophic factors are generallymediated through binding to cell surface receptors. The receptors for anumber of these factors have been identified and antibodies andmolecular probes for specific receptors are available. Neural stem cellscan be analyzed for the presence of growth factor receptors at allstages of differentiation. In many cases, the identification of aparticular receptor will define the strategy to use in furtherdifferentiating the cells along specific developmental pathways with theaddition of exogenous growth or trophic factors.

Exogenous growth factors can be added alone or in various combinations.They can also be added in a temporal sequence (i.e. exposure to a firstgrowth factor influences the expression of a second growth factorreceptor, Neuron 4:189-201 (1990). Among the growth factors and othermolecules that can be used to influence the differentiation of precursorcells in vitro are FGF-1, FGF-2, ciliary neurotrophic factor (CNTF),NGF, brain derived neurotrophic factor (BDNF), neurotrophin 3,neurotrophin 4, interleukins, leukemia inhibitory factor (LIF), cyclicadenosine monophosphate, forskolin, tetanus toxin, high levels ofpotassium, amphiregulin, TGF-α, TGF-β, insulin-like growth factors,dexamethasone (glucocorticoid hormone), isobutyl 3-methylxanthine,somatostatin, growth hormone, retinoic acid, and PDGF. These and othergrowth factors and molecules will find use in the present invention.

Genetic Modification of Neural Stem Cell Progeny

Although the precursor cells are non-transformed primary cells, theypossess features of a continuous cell line. In the undifferentiatedstate, in the presence of a proliferation-inducing growth factor such asEGF, the cells continuously divide and are therefore excellent targetsfor genetic modification. The term "genetic modification" as used hereinrefers to the stable or transient alteration of the genotype of aprecursor cell by intentional introduction of exogenous DNA. DNA may besynthetic, or naturally derived, and may contain genes, portions ofgenes, or other useful DNA sequences. The term "genetic modification" asused herein is not meant to include naturally occurring alterations suchas that which occurs through natural viral activity, natural geneticrecombination, or the like.

Exogenous DNA may be introduced to a precursor cell by viral vectors(retrovirus, modified herpes viral, herpes-viral, adenovirus,adeno-associated virus, and the like) or direct DNA transfection(lipofection, calcium phosphate transfection, DEAE-dextran,electroporation, and the like). The genetically modified cells of thepresent invention possess the added advantage of having the capacity tofully differentiate to produce neurons or macroglial cells in areproducible fashion using a number of differentiation protocols.

In another embodiment, the precursor cells are derived from transgenicanimals, and thus are in a sense already genetically modified. There areseveral methods presently used for generating transgenic animals. Thetechnique used most often is direct microinjection of DNA intosingle-celled fertilized eggs. Other techniques includeretroviral-mediated transfer, or gene transfer in embryonic stem cells.

These techniques and others are detailed by Hogan et al. in Manipulatingthe Mouse Embryo, A Laboratory Manual (Cold Spring Harbor LaboratoryEd., 1986). Use of these transgenic animals has certain advantagesincluding the fact that there is no need to transfect healthyneurospheres. Precursor cells derived from transgenic animals willexhibit stable gene expression. Using transgenic animals, it is possibleto breed in new genetic combinations. The transgenic animal may haveintegrated into its genome any useful gene that is expressed by neuralcells. Examples of useful DNA are given below in the discussion ofgenetically modifying precursor cells.

A significant challenge for cellular transplantation in the CNS is theidentification of the donor cells after implantation within the host. Anumber of strategies have been employed to mark donor cells, includingtritiated labels, fluorescent dyes, dextrans, and viral vectors carryingreporter genes. However, these methods suffer from inherent problems oftoxicity, stability, or dilution over the long term. The use of neuralcells derived from transgenic animals may provide an improved means bywhich identification of transplanted neural cells can be achieved. Atransgenic marking system provides a more stable and efficient methodfor cell labeling. In this system, promoter elements, for example forGFAP and MBP, can direct the expression of the E. coli B-galactosidasereporter gene in transgenic mice. In these systems, cell-specificexpression of the reporter gene occurs in astrocytes (GFAP-lacZ) and inoligodendrocytes MBP-lacZ) in a developmentally-regulated manner. TheRosa26 transgenic mice, described in Example 45, is one example of atransgenic marking system in which all cells ubiquitously expressβ-galactosidase.

Once propagated, the neurosphere cells are mechanically dissociated intoa single cell suspension and plated on petri dishes in a medium wherethey are allowed to attach overnight. The precursor cells are thengenetically modified. If the precursor cells are generated fromtransgenic animals, then they may or may not be subjected to furthergenetic modification, depending upon the properties desired of thecells. Any useful genetic modification of the cells is within the scopeof the present invention. For example, precursor cells may be modifiedto produce or increase production of a biologically active substancesuch as a neurotransmitter or growth factor or the like. The geneticmodification is performed either by infection with recombinantretroviruses or transfection using methods known in the art (seeManiatis et al., in Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, N.Y. (1982)). Briefly, the chimeric gene constructswill contain viral, for example retroviral long terminal repeat (LTR),simian virus 40 (SV40), cytomegalovirus (CMV); or mammaliancell-specific promoters such as tyrosine hydroxylase (TH, a marker fordopamine cells), DBH, phenylethanolamine N-methyltransferase (PNMT),ChAT, GFAP, NSE, the NF proteins (NF-L, NF-M, NF-H, and the like) thatdirect the expression of the structural genes encoding the desiredprotein. In addition, the vectors will include a drug selection marker,such as the E. coli aminoglycoside phosphotransferase gene, which whencoinfected with the experimental gene confers resistance to geneticin(G418), a protein synthesis inhibitor.

When the genetic modification is for the production of a biologicallyactive substance, the substance will generally be one that is useful forthe treatment of a given CNS disorder. For example, it may be desired togenetically modify cells so they secrete a certain growth factorproduct. As used herein, the term "growth factor product" refers to aprotein, peptide, mitogen, or other molecule having a growth,proliferative, differentiative, or trophic effect. Growth factorproducts useful in the treatment of CNS disorders include, but are notlimited to, NGF, BDNF, the neurotrophins (NT-3, NT-4/NT-5), CNTF,amphiregulin, FGF-1, FGF-2, EGF, TGFα, TGFβs, PDGF, IGFs, and theinterleukins.

Cells can also be modified to express a certain growth factor receptor(r) including, but not limited to, p75 low affinity NGFr, CNTFr, the trkfamily of neurotrophin receptors (trk, trkB, trkC), EGFr, FGFr, andamphiregulin receptors. Cells can be engineered to produce variousneurotransmitters or their receptors such as serotonin, L-dopa,dopamine, norepinephrine, epinephrine, tachykinin, substance-P,endorphin, enkephalin, histamine, N-methyl D-aspartate, glycine,glutamate, GABA, ACh, and the like. Useful neurotransmitter-synthesizinggenes include TH, DDC, DBH, PNMT, GAD, tryptophan hydroxylase, ChAT, andhistidine decarboxylase. Genes that encode for various neuropeptides,which may prove useful in the treatment of CNS disorders, includesubstance-P, neuropeptide-Y, enkephalin, vasopressin, VIP, glucagon,bombesin, CCK, somatostatin, calcitonin gene-related peptide, and thelike.

After successfully transfected/infected cells are selected they can becloned using limiting dilution in 96 multi-well plates and assayed forthe presence of the desired biologically active substance. Clones thatexpress high levels of the desired substance are grown and their numbersexpanded in T-flasks. The specific cell line can then be cyropreserved.Multiple clones of genetically modified precursor cells will beobtained. Some may give rise preferentially to neuronal cells, and someto glial cells.

The genetically modified precursor cells can be implanted for cell/genetherapy into the CNS of a recipient in need of the biologically activemolecule produced by the genetically modified cells. Transplantationtechniques are detailed below. Alternatively, the genetically modifiedprecursor cells can be subjected to various differentiation protocols invitro prior to implantation. For example, genetically modified precursorcells may be removed from the culture medium which allows proliferationand differentiated using any of the protocols described above. Theprotocol used will depend upon the type of genetically modified celldesired. Once the cells have differentiated, they are again assayed forexpression of the desired protein. Cells having the desired phenotypecan be isolated and implanted into recipients in need of the protein orbiologically active molecule that is expressed by the geneticallymodified cell.

Transplantation of Neural Stem Cell Progeny Alleviate Disorders of theCNS in Animal Models Caused by Disease or Injury

It is well recognized in the art that transplantation of tissue into theCNS offers the potential for treatment of neurodegenerative disordersand CNS damage due to injury (review: Lindvall, (1991) Tins vol. 14(8):376-383). Transplantation of new cells into the damaged CNS has thepotential to repair damaged circuitries and provide neurotransmittersthereby restoring neurological function. However, the absence ofsuitable cells for transplantation purposes has prevented the fullpotential of this procedure from being met. "Suitable" cells are cellsthat meet the following criteria: 1) can be obtained in large numbers;2) can be proliferated in vitro to allow insertion of genetic material,if necessary; 3) capable of surviving indefinitely but stop growingafter transplantation to the brain; 4) are nonimmunogenic, preferablyobtained from a patient's own tissue; 5) are able to form normal neuralconnections and respond to neural physiological signals (Bjorklund(1991) TINS Vol. 14(8): 319-322). The progeny of multipotent neural stemcells obtainable from embryonic or adult CNS tissue, which are able todivide indefinitely when maintained in vitro using the cultureconditions described herein, meet all of the desirable requirements ofcells suitable for neural transplantation purposes and are aparticularly suitable cell line as the cells have not been immortalizedand are not of tumorigenic origin. The use of multipotent neural stemcells in the treatment of neurological disorders and CNS damage can bedemonstrated by the use of animal models.

The neural stem cell progeny can be administered to any animal withabnormal neurological or neurodegenerative symptoms obtained in anymanner, including those obtained as a result of mechanical, chemical, orelectrolytic lesions, as a result of experimental aspiration of neuralareas, or as a result of aging processes. Particularly preferablelesions in non-human animal models are obtained with 6-hydroxy-dopamine(6-OHDA), 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), ibotenicacid and the like.

The instant invention allows the use of precursor cells prepared fromdonor tissue which is xenogeneic to the host. Since the CNS is asomewhat immunoprivileged site, the immune response is significantlyless to xenografts, than elsewhere in the body. In general, however, inorder for xenografts to be successful it is preferred that some methodof reducing or eliminating the immune response to the implanted tissuebe employed. Thus recipients will often be immunosuppressed, eitherthrough the use of immunosuppressive drugs such as cyclosporin, orthrough local immunosuppression strategies employing locally appliedimmunosuppressants.

Local immunosuppression is disclosed by Gruber, Transplantation 54:1-11(1992).

Rossini, U.S. Pat. No. 5,026,365, discloses encapsulation methodssuitable for local immunosuppression.

As an alternative to employing immunosuppression techniques, methods ofgene replacement or knockout using homologous recombination in embryonicstem cells, taught by Smithies et al. (Nature, 317:230-234 (1985), andextended to gene replacement or knockout in cell lines (H. Zheng 35 al.,PNAS, 88:8067-8071 (1991)), can be applied to precursor cells for theablation of major histocompatibility complex (MHC) genes. Precursorcells lacking MHC expression would allow for the grafting of enrichedneural cell populations across allogeneic, and perhaps even xenogeneic,histocompatibility barriers without the need to immunosuppress therecipient. General reviews and citations for the use of recombinantmethods to reduce antigenicity of donor cells are also disclosed byGruber (supra). Exemplary approaches to the reduction of immunogenicityof transplants by surface modification are disclosed by Faustman WO92/04033 (1992). Alternatively the immunogenicity of the graft may bereduced by preparing precursor cells from a transgenic animal that hasaltered or deleted MHC antigens.

Grafting of precursor cells prepared from tissue which is allogeneic tothat of the recipient will most often employ tissue typing in an effortto most closely match the histocompatibility type of the recipient.Donor cell age as well as age of the recipient have been demonstrated tobe important factors in improving the probability of neuronal graftsurvival. The efficiency of grafting is reduced with increased age ofdonor cells. Furthermore, grafts are more readily accepted by youngerrecipients compared to older recipients. These two factors are likely tobe as important for glial graft survival as they are for neuronal graftsurvival.

In some instances, it may be possible to prepare neural stem cellprogeny from the recipient's own nervous system (e.g.in the case oftumor removal biopsies etc,). In such instances the neural stem cellprogeny may be generated from dissociated tissue and proliferated invitro using the methods described above. Upon suitable expansion of cellnumbers, the precursor cells may be harvested, genetically modified ifnecessary, and readied for direct injection into the recipient's CNS.

Transplantation can be done bilaterally, or, in the case of a patientsuffering from Parkinson's Disease, contralateral to the most affectedside. Surgery is performed in a manner in which particular brain regionsmay be located, such as in relation to skull sutures, particularly witha stereotaxic guide. Cells are delivered throughout any affected neuralarea, in particular to the basal ganglia, and preferably to the caudateand putamen, the nucleus basalis or the substantia nigra. Cells areadministered to the particular region using any method which maintainsthe integrity of surrounding areas of the brain, preferably by injectioncannula. Injection methods exemplified by those used by Duncan et al.J.Neurocytology, 17:351-361 (1988), and scaled up and modified for usein humans are preferred. Methods taught by Gage et al., supra, for theinjection of cell suspensions such as fibroblasts into the CNS may alsobe employed for injection of neural precursor cells. Additionalapproaches and methods may be found in Neural Grafting in the MammalianCNS, Bjorklund and Stenevi, eds., (1985).

Although solid tissue fragments and cell suspensions of neural tissueare immunogenic as a whole, it could be possible that individual celltypes within the graft are themselves immunogenic to a lesser degree.For example, Bartlett et al. (Prog. Brain Res. 82: 153-160 (1990)) haveabrogated neural allograft rejection by pre-selecting a subpopulation ofembryonic neuroepithelial cells for grafting by the use of immunobeadseparation on the basis of MHC expression. Thus, another approach isprovided to reduce the chances of allo and xenograft rejection by therecipient without the use of immunosuppression techniques.

Neural stem cell progeny when administered to the particular neuralregion preferably form a neural graft, wherein the neuronal cells formnormal neuronal or synaptic connections with neighboring neurons, andmaintain contact with transplanted or existing glial cells which mayform myelin sheaths around the neurons' axons, and provide a trophicinfluence for the neurons. As these transplanted cells form connections,they re-establish the neuronal networks which have been damaged due todisease and aging.

Survival of the graft in the living host can be examined using variousnon-invasive scans such as computerized axial tomography (CAT scan or CTscan), nuclear magnetic resonance or magnetic resonance imaging (NMR orMRI) or more preferably positron emission tomography (PET) scans.Post-mortem examination of graft survival can be done by removing theneural tissue, and examining the affected region macroscopically, ormore preferably using microscopy. Cells can be stained with any stainsvisible under light or electron microscopic conditions, moreparticularly with stains which are specific for neurons and glia.Particularly useful are monoclonal antibodies which identify neuronalcell surface markers such as the M6 antibody which identifies mouseneurons. Most preferable are antibodies which identify anyneurotransmitters, particularly those directed to GABA, TH, ChAT, andsubstance P, and to enzymes involved in the synthesis ofneurotransmitters, in particular, GAD. Transplanted cells can also beidentified by prior incorporation of tracer dyes such as rhodamine- orfluorescein-labelled microspheres, fast blue, bisbenzamide orretrovirally introduced histochemical markers such as the lac Z genewhich produces beta galactosidase.

Functional integration of the graft into the host's neural tissue can beassessed by examining the effectiveness of grafts on restoring variousfunctions, including but not limited to tests for endocrine, motor,cognitive and sensory functions. Motor tests which can be used includethose which quantitate rotational movement away from the degeneratedside of the brain, and those which quantitate slowness of movement,balance, coordination, akinesia or lack of movement, rigidity andtremors. Cognitive tests include various tests of ability to performeveryday tasks, as well as various memory tests, including mazeperformance.

Neural stem cell progeny can be produced and transplanted using theabove procedures to treat demyelination diseases. Human demyelinatingdiseases for which the cells of the present invention may providetreatment include disseminated perivenous encephalomyelitis, MS (Charcotand Marburg types), neuromyelitis optica, concentric sclerosis, acute,disseminated encephalomyelitides, post encephalomyelitis, postvaccinalencephalomyelitis, acute hemorrhagic leukoencephalopathy, progressivemultifocal leukoencephalopathy, idiopathic polyneuritis, diphthericneuropathy, Pelizaeus-Merzbacher disease, neuromyelitis optica, diffusecerebral sclerosis, central pontine myelinosis, spongiformleukodystrophy, and leukodystrophy (Alexander type).

Areas of demyelination in humans is generally associated with plaquelike structures. Plaques can be visualized by magnetic resonanceimaging. Accessible plaques are the target area for injection of neuralstem cell progeny. Standard stereotactic neurosurgical methods are usedto inject cell suspensions both into the brain and spinal cord.Generally, the cells can be obtained from any of the sources discussedabove. However, in the case of demyelinating diseases with a geneticbasis directly affecting the ability of the myelin forming cell tomyelinate axons, allogeneic tissue would be a preferred source of thecells as autologous tissue (i.e. the recipient's cells) would generallynot be useful unless the cells have been modified in some way to insurethe lesion will not continue (e.g. genetically modifying the cells tocure the demyelination lesion).

Oligodendrocytes derived from neural stem cell progeny proliferated anddifferentiated in vitro may be injected into demyelinated target areasin the recipient. Appropriate amounts of type I astrocytes may also beinjected. Type I astrocytes are known to secrete PDGF which promotesboth migration and cell division of oligodendrocytes. [Nobel et al.,Nature 333:560-652 (1988); Richardson et al., Cell, 53:309-319 (1988)].

A preferred treatment of demyelination disease uses undifferentiatedneural stem cell progeny. Neurospheres grown in the presence of aproliferation-inducing growth factor such as EGF can be dissociated toobtain individual precursor cells which are then placed in injectionmedium and injected directly into the demyelinated target region. Thecells differentiate in vivo. Astrocytes can promote remyelination invarious paradigms. Therefore, in instances where oligodendrocyteproliferation is important, the ability of precursor cells to give riseto type I astrocytes may be useful. In other situations, PDGF may beapplied topically during the transplantation as well as with repeateddoses to the implant site thereafter.

The injection of neural stem cell progeny in remyelination therapyprovides, amongst other types of cells, a source of immature type Iastrocytes at the implant site. This is a significant feature becauseimmature astrocytes (as opposed to mature astrocytes) have a number ofspecific characteristics that make them particularly suited forremyelination therapy. First, immature, as opposed to mature, type Iastrocytes are known to migrate away from the implant site [Lindsay et.al, Neurosci. 12:513-530 (1984)] when implanted into a mature recipientand become associated with blood vessels in the recipient's CNS [Silveret al., WO 91/06631 (1991)]. This is at least partially due to the factthat immature astrocytes are intrinsically more motile than matureastrocytes. [Duffy et al., Exp Cell Res. 139:145-157 (1982), Table VII].Type I astrocytes differentiating at or near the precursor cell implantsite should have maximal motility and thereby optimize the opportunityfor oligodendrocyte growth and division at sites distant from theimplant. The localization of the astrocytes near blood vessels is alsosignificant from a therapeutic standpoint since (at least in MS) mostplaques have a close anatomical relationship with one or more veins.Another characteristic of immature astrocytes that makes themparticularly suited for remyelination therapy is that they undergo alesser degree of cell death than mature type I astrocytes. (Silver etal., supra)

Any suitable method for the implantation of precursor cells near to thedemyelinated targets may be used so that the cells can become associatedwith the demyelinated axons. Glial cells are motile and are known tomigrate to, along, and across their neuronal targets thereby allowingthe spacing of injections. Remyelination by the injection of precursorcells is a useful therapeutic in a wide range of demyelinatingconditions. It should also be borne in mind that in some circumstancesremyelination by precursor cells will not result in permanentremyelination, and repeated injections will be required. Suchtherapeutic approaches offer advantage over leaving the conditionuntreated and may spare the recipient's life.

In Vivo Proliferation, Differentiation, and Genetic Modification ofNeural Stem Cell Progeny

Neural stem cells and their progeny can be induced to proliferate anddifferentiate in vivo by administering to the host, any growth factor(s)or pharmaceutical composition that will induce proliferation anddifferentiation of the cells. These growth factors include any growthfactor known in the art, including the growth factors described abovefor in vitro proliferation and differentiation. Pharmaceuticalcompositions include any substance that blocks the inhibitory influenceand/or stimulates neural stem cells and stem cell progeny to proliferateand ultimately differentiate. Thus, the techniques described above toproliferate, differentiate, and genetically modify neural stem cells invitro can be adapted to in vivo techniques, to achieve similar results.Such in vivo manipulation and modification of these cells allows cellslost, due to injury or disease, to be endogenously replaced, thusobviating the need for transplanting foreign cells into a patient.Additionally, the cells can be modified or genetically engineered invivo so that they express various biological agents useful in thetreatment of neurological disorders.

Administration of growth factors can be done by any method, includinginjection cannula, transfection of cells with growth hormone-expressingvectors, injection, timed-release apparati which can administersubstances at the desired site, and the like. Pharmaceuticalcompositions can be administered by any method, including injectioncannula, injection, oral administration, timed-release apparati and thelike. The neural stem cells can be induced to proliferate anddifferentiate in vivo by induction with particular growth factors orpharmaceutical compositions which will induce their proliferation anddifferentiation. Therefore, this latter method circumvents the problemsassociated with transplantation and immune reactions to foreign cells.Any growth factor can be used, particularly EGF, TGFα, FGF-1, FGF-2 andNGF.

Growth factors can be administered in any manner known in the art inwhich the factors may either pass through or by-pass the blood-brainbarrier. Methods for allowing factors to pass through the blood-brainbarrier include minimizing the size of the factor, or providinghydrophobic factors which may pass through more easily.

The fact that neural stem cells are located in the tissues liningventricles of mature brains offers several advantages for themodification and manipulation of these cells in vivo and the ultimatetreatment of various neurological diseases, disorders, and injury thataffect different regions of the CNS. Therapy for these can be tailoredaccordingly so that stem cells surrounding ventricles near the affectedregion would be manipulated or modified in vivo using the methodsdescribed herein. The ventricular system is found in nearly all brainregions and thus allows easier access to the affected areas. If onewants to modify the stem cells in vivo by exposing them to a compositioncomprising a growth factor or a viral vector, it is relatively easy toimplant a device that administers the composition to the ventricle andthus, to the neural stem cells. For example, a cannula attached to anosmotic pump may be used to deliver the composition. Alternatively, thecomposition may be injected directly into the ventricles. The neuralstem cell progeny can migrate into regions that have been damaged as aresult of injury or disease. Furthermore, the close proximity of theventricles to many brain regions would allow for the diffusion of asecreted neurological agent by the stem cells or their progeny.

For treatment of Huntington's Disease, Alzheimer's Disease, Parkinson'sDisease, and other neurological disorders affecting primarily theforebrain, growth factors or other neurological agents would bedelivered to the ventricles of the forebrain to affect in vivomodification or manipulation of the stem cells. For example, Parkinson'sDisease is the result of low levels of dopamine in the brain,particularly the striatum. It would be advantageous to induce apatient's own quiescent stem cells to begin to divide in vivo and toinduce the progeny of these cells to differentiate into dopaminergiccells in the affected region of the striatum, thus locally raising thelevels of dopamine.

Normally the cell bodies of dopaminergic neurons are located in thesubstantia nigra and adjacent regions of the mesencephalon, with theaxons projecting to the striatum. Prior art methods for treatingParkinson's disease usually involves the use of the drug L-Dopa, toraise dopamine levels in the striatum. However, there are disadvantageswith this treatment including drug tolerance and side effects. Also,embryonic tissues that produce dopamine have been transplanted into thestriatum of human Parkinsonian patients with reasonable success.However, the use of large quantities of fetal human tissue required forthis procedure raises serious ethical concerns and practical issues.

The methods and compositions of the present invention provide analternative to the use of drugs and the controversial use of largequantities of embryonic tissue for treatment of Parkinson's disease.Dopamine cells can be generated in the striatum by the administration ofa composition comprising growth factors to the lateral ventricle. Aparticularly preferred composition comprises a combination of EGF,FGF-2, and heparan sulphate. The composition preferably also comprisesserum. After administration of this composition, there is a significantincrease in the transcription of messenger RNA (mRNA) for TH in thesubventricular region of the striatum, an area which normally does notcontain dopaminergic cell bodies. These methods and results aredescribed in detail in Example 34. As detailed in Example 35, the use ofdual labeling tissue to show the distribution of BrdU+ and TH+ cellsindicates that, in response to the in vivo administration of growthfactors, TH+ cell bodies occur in striatal tissue. Many of these newlygenerated TH+ cells are also BrdU+.

For the treatment of MS and other demyelinating or hypomyelinatingdisorders, and for the treatment of Amyotrophic Lateral Sclerosis orother motor neuron diseases, growth factors or other neurological agentswould be delivered to the central canal.

In addition to treating CNS tissue immediately surrounding a ventricle,a viral vector, DNA, growth factor, or other neurological agent can beeasily administered to the lumbar cistern for circulation throughout theCNS.

Under normal conditions subependymal precursors do not differentiate ormigrate, rather, their fate appears to be cell death after an undefinednumber of cell divisions (Morshead and Van der Kooy, supra). Thisexplanation is also supported by PCR evidence, as described above.Injection of growth factors into the lateral ventricle alters this fate.As described in more detail in Example 27 below, retroviruses wereinjected into the lateral ventricles for six consecutive days.Implanting cannulae attached to EGF-filled osmotic pumps into thelateral ventricles on the same day as (and 1 or 6 days following)retrovirus injection results in an increase in the total number ofRV-β-gal labelled cells 6 days later (from an average of 20 cells/brainto 150 cells/brain).

It is known from the PCR experiments described above that 6 daysfollowing retroviral injection no cells exist that contain non-expressedretroviral DNA. Thus these results indicate that the EGF-inducedincrease in β-gal positive cell number is due to the expansion of theclone size of the retrovirally labelled constitutively proliferativepopulation. It is also possible that part of this increase is due to theactivation by EGF of a relatively quiescent stem cell.

Interestingly, this expansion of the number of B-gal labelled cells isaccompanied by the migration of these cells away from the subependymalmedially, laterally, rostrally, and caudally with subsequentdifferentiation. Thus, infusion of EGF or similar growth factors inducesthe proliferation, migration and differentiation of neural stem cellsand progenitor cells in vivo, and can be used therapeutically to replaceneural cells lost due to injury or disease. In a preferred embodimentEGF and FGF are administered together or sequentially.

The normal fate of the constitutively proliferating cell population(i.e. cell death) can be altered by administering Bcl-2 or geneticallymodifying the cells with the bcl-2 gene. The gene product is known toprevent programmed cell death (apoptosis) in a variety of cell types.Similar to the EGF experiments, a clonal expansion of the constitutivelyproliferating cell population is achieved following infection withbcl-2.

Other ways of passing the blood-brain barrier include in vivotransfection of neural stem cells and stem cell progeny with expressionvectors containing genes that code for growth factors, so that the cellsthemselves produce the factor. Any useful genetic modification of thecells is within the scope of the present invention. For example, inaddition to genetic modification of the cells to express growth factors,the cells may be modified to express other types of neurological agentssuch as neurotransmitters. Preferably, the genetic modification isperformed either by infection of the cells lining ventricular regionswith recombinant retroviruses or transfection using methods known in theart including CaPO₄ transfection, DEAE-dextran transfection, polybrenetransfection, by protoplast fusion, electroporation, lipofection, andthe like [see Maniatis et al., supra]. Any method of geneticmodification, now known or later developed can be used. With direct DNAtransfection, cells could be modified by particle bombardment, receptormediated delivery, and cationic liposomes. When chimeric gene constructsare used, they generally will contain viral, for example retroviral longterminal repeat (LTR), simian virus 40 (SV40), cytomegalovirus (CMV); ormammalian cell-specific promoters such as those for TH, DBH,phenylethanolamine N-methyltransferase, ChAT, GFAP, NSE, the NF proteins(NF-L, NF-M, NF-H, and the like) that direct the expression of thestructural genes encoding the desired protein.

If a retroviral construct is to be used to genetically modify normallyquiescent stem cells, then it is preferable to induce the proliferationof these cells using the methods described herein. For example, anosmotic infusion pump could be used to deliver growth factors to thecentral canal several days prior to infection with the retrovirus. Thisassures that there will be actively dividing neural stem cells which aresusceptible to infection with the retrovirus.

When the genetic modification is for the production of a biologicallyactive substance, the substance will generally be one that is useful forthe treatment of a given CNS disorder. For example, it may be desired togenetically modify cells so they secrete a certain growth factorproduct. Growth factor products useful in the treatment of CNS disordersare listed above. Cells can also be modified in vivo to express a growthfactor receptors, neurotransmitters or their receptors,neurotransmitter-synthesizing genes, neuropeptides, and the like, asdiscussed above.

Any expression vector known in the art can be used to express the growthfactor, as long as it has a promoter which is active in the cell, andappropriate termination and polyadenylation signals. These expressionvectors include recombinant vaccinia virus vectors including pSC11, orvectors derived various viruses such as from Simian Virus 40 (SV40, i.e.pSV2-dhfr, pSV2neo, pko-neo, pSV2gpt, pSVT7 and pBABY), from RousSarcoma Virus (RSV, i.e. pRSVneo), from mouse mammary tumor virus (MMTV,i.e. pMSG), from adenovirus(pMT2), from herpes simplex virus (HSV, i.e.pTK2 and pHyg), from bovine papillomavirus (BPV, i.e. pdBPV andpBV-1MTHA), from Epstein-Barr Virus (EBV, i.e. p205 and pHEBo) or anyother eukaryotic expression vector known in the art.

Other methods for providing growth factors to the area oftransplantation include the implantation into the brain in proximity tothe graft of any device which can provide an infusion of the factor tothe surrounding cells.

In Vitro Models of CNS Development, Function and Dysfunction, andMethods for Screening Effects of Drugs on Neural Cells

Neural stem cell progeny cultured in vitro can be used for the screeningof potential neurologically therapeutic compositions. These compositionscan be applied to cells in culture at varying dosages, and the responseof the cells monitored for various time periods. Physicalcharacteristics of the cells can be analyzed by observing cell andneurite growth with microscopy. The induction of expression of new orincreased levels of proteins such as enzymes, receptors and other cellsurface molecules, or of neurotransmitters, amino acids, neuropeptidesand biogenic amines can be analyzed with any technique known in the artwhich can identify the alteration of the level of such molecules. Thesetechniques include immunohistochemistry using antibodies against suchmolecules, or biochemical analysis. Such biochemical analysis includesprotein assays, enzymatic assays, receptor binding assays, enzyme-linkedimmunosorbant assays (ELISA), electrophoretic analysis, analysis withhigh performance liquid chromatography (HPLC), Western blots, andradioimmune assays (RIA). Nucleic acid analysis such as Northern blotscan be used to examine the levels of mRNA coding for these molecules, orfor enzymes which synthesize these molecules.

Alternatively, cells treated with these pharmaceutical compositions canbe transplanted into an animal, and their survival, ability to formneuronal connections, and biochemical and immunological characteristicsexamined as previously described.

For the preparation of CNS models, neural stem cells and stem cellprogeny are proliferated using the methods described above. Upon removalof the proliferation-inducing growth factor, proliferation ofmultipotent neural stem cells ceases. The neurospheres can bedifferentiated using the methods described above, for example byadhering the neurospheres to a substrate such as poly-ornithine-treatedplastic or glass where the precursor cells begin to differentiate intoneurons and glial cells. Thus, the proliferation-inducing growth factoracts as an extrinsic signaling molecule that can be added or removed atwill to control the extent of proliferation.

When the proliferation-inducing growth factor is removed, thegrowth-factor responsive stem cell progeny can be co-cultured on afeeder layer. Many types of feeder layers may be used, such asfibroblasts, neurons, astrocytes, oligodendrocytes, tumor cell lines,genetically altered cell lines or any cells or substrate with bioactiveproperties. The feeder layer generally produces a broader range ofphenotypes. In this instance, the feeder layer acts as a substrate andsource of both membrane bound and soluble factors that induce and alterthe differentiation of the stem cell-generated progeny. Compared to amore inert substance, such as poly-L-omithine, an astrocyte feederlayer, for example, induces a broader range of neuronal phenotypes asdetermined by indirect immunocytochemistry at 7 DIV. When differentiatedon a poly-L-ornithine coated substrate with 1% FBS, neuronal phenotypesare almost exclusively GABAergic or substance P-ergic. Whendifferentiated on an astrocyte feeder layer, in addition to GABAergicand substance P-ergic neurons, somatostatin, neuropeptide Y (NPY),glutamate and met-enkephalin-containing neurons are present. Theastrocytes can be derived from tissue obtained from various brainregions such as the striatum, cortex and spinal cord.

Once the growth factor is removed, the culture medium may contain serumsuch as 0.5-1.0% FBS. Serum tends to support the differentiation processand enhance cell survival, especially when the differentiating cells aregrown at a low density. However, it is possible to culture anddifferentiate the cells using defined conditions.

Within 1-3 days after removal of the growth factor and placing of thecell in conditions that support differentiation and survival, most orall of the precursor cells begin to lose immunoreactivity for nestin andbegin to express antigens specific for neurons, astrocytes oroligodendrocytes. The identification of neurons is confirmed usingimmunoreactivity for the neuron-specific markers previously mentioned.

The precursor cells described above can be used in methods ofdetermining the effect of a biological agents on neural cells. The term"biological agent" refers to any agent, such as a virus, protein,peptide, amino acid, lipid, carbohydrate, nucleic acid, nucleotide,drug, pro-drug or other substance that may have an effect on neuralcells whether such effect is harmful, beneficial, or otherwise.Biological agents that are beneficial to neural cells are referred toherein as "neurological agents", a term which encompasses anybiologically or pharmaceutically active substance that may provepotentially useful for the proliferation, differentiation or functioningof CNS cells or treatment of neurological disease or disorder. Forexample, the term may encompass certain neurotransmitters,neurotransmitter receptors, growth factors, growth factor receptors, andthe like, as well as enzymes used in the synthesis of these agents.

Examples of biological agents include growth factors such as FGF-1,FGF-2, EGF and EGF-like ligands, TGFα, IGF-1, NGF, PDGF, and TGFβs;trophic factors such as BDNF, CNTF, and glial-derived neurotrophicfactor (GDNF); regulators of intracellular pathways associated withgrowth factor activity such as phorbol 12-myristate 13-acetate,staurosporine, CGP-41251, tyrphostin, and the like; hormones such asactivin and TRH; various proteins and polypeptides such as interleukins,the Bcl-2 gene product, bone morphogenic protein (BMP-2), macrophageinflammatory proteins (MIP-1α, MIP-1β and MIP-2); oligonucleotides suchas antisense strands directed, for example, against transcripts for EGFreceptors, FGF receptors, and the like; heparin-like molecules such asheparan sulfate; and a variety of other molecules that have an effect onneural stem cells or stem cell progeny including amphiregulin, retinoicacid, and tumor necrosis factor alpha (TNFα).

To determine the effect of a potential biological agent on neural cells,a culture of precursor cells derived from multipotent stem cells can beobtained from normal neural tissue or, alternatively, from a hostafflicted with a CNS disease or disorder such as Alzheimer's Disease,Parkinson's Disease, or Down's Syndrome. The choice of culture willdepend upon the particular agent being tested and the effects one wishesto achieve. Once the cells are obtained from the desired donor tissue,they are proliferated in vitro in the presence of aproliferation-inducing growth factor.

The ability of various biological agents to increase, decrease or modifyin some other way the number and nature of the stem cell progenyproliferated in the presence of EGF or other proliferative factor can bescreened on cells proliferated by the methods described in Examples 1-6.For example, it is possible to screen for biological agents thatincrease the proliferative ability of progenitor cells which would beuseful for generating large numbers of cells for transplantationpurposes. It is also possible to screen for biological agents whichinhibit precursor cell proliferation. In these studies precursor cellsare plated in the presence of the biological factor(s) of interest andassayed for the degree of proliferation which occurs. The effects of abiological agent or combination of biological agents on thedifferentiation and survival of progenitor cells and their progeny canbe determined. It is possible to screen neural cells which have alreadybeen induced to differentiate prior to the screening. It is alsopossible to determine the effects of the biological agents on thedifferentiation process by applying them to precursor cells prior todifferentiation. Generally, the biological agent will be solubilized andadded to the culture medium at varying concentrations to determine theeffect of the agent at each dose. The culture medium may be replenishedwith the biological agent every couple of days in amounts so as to keepthe concentration of the agent somewhat constant.

Changes in proliferation are observed by an increase or decrease in thenumber of neurospheres that form and/or an increase or decrease in thesize of the neurospheres (which is a reflection of the rate ofproliferation--determined by the numbers of precursor cells perneurosphere). Thus, the term "regulatory factor" is used herein to referto a biological factor that has a regulatory effect on the proliferationof stem cells and/or precursor cells. For example, a biological factorwould be considered a "regulatory factor" if it increases or decreasesthe number of stem cells that proliferate in vitro in response to aproliferation-inducing growth factor (such as EGF). Alternatively, thenumber of stem cells that respond to proliferation-inducing factors mayremain the same, but addition of the regulatory factor affects the rateat which the stem cell and stem cell progeny proliferate. Aproliferative factor may act as a regulatory factor when used incombination with another proliferative factor. For example, theneurospheres that form in the presence of a combination of bFGF and EGFare significantly larger than the neurospheres that form in the presenceof bFGF alone, indicating that the rate of proliferation of stem cellsand stem cell progeny is higher.

Other examples of regulatory factors include heparan sulfate, TGFβs,activin, BMP-2, CNTF, retinoic acid, TNFα, MIP-1α, MIP-1β, MIP-2, NGF,PDGF, interleukins, and the Bcl-2 gene product. Antisense molecules thatbind to transcripts of proliferative factors and the transcripts fortheir receptors also regulate stem cell proliferation. Other factorshaving a regulatory effect on stem cell proliferation include those thatinterfere with the activation of the c-fos pathway (an intermediateearly gene, known to be activated by EGF), including phorbol 12myristate 13-acetate (PMA; Sigma), which up-regulates the c-fos pathwayand staurosporine (Research Biochemical International) and CGP-41251(Ciba-Geigy), which down regulate c-fos expression and factors, such astyrphostin [Fallon, D et al., Mol. Cell Biol., 11 (5): 2697-2703 (1991)]and the like, which suppress tyrosine kinase activation induced by thebinding of EGF to its receptor.

Preferred regulatory factors for increasing the rate at which neuralstem cell progeny proliferate in response to FGF are heparan sulfate andEGF. Preferred regulatory factors for decreasing the number of stemcells that respond to proliferative factors are members of the TGFBfamily, interleukins, MIPs, PDGF, TNFα, retinoic acid (10⁻⁶ M) and CNTF.Preferred factors for decreasing the size of neurospheres generated bythe proliferative factors are members of the TGFB family, retinoic acid(10⁻⁶ M) and CNTF.

The regulatory factors are added to the culture medium at aconcentration in the range of about 10 pg/ml to 500 ng/ml, preferablyabout 1 ng/ml to 100 ng/ml. The most preferred concentration forregulatory factors is about 10 ng/ml. The regulatory factor retinoicacid is prepared from a 1 mM stock solution and used at a finalconcentration between about 0.01 μM and 100 μM, preferably between about0.05 to 5 μM. Preferred for reducing the proliferative effects of EGF orbFGF on neurosphere generation is a concentration of about 1 μM ofretinoic acid. Antisense strands, can be used at concentrations fromabout 1 to 25 μM. Preferred is a range of about 2 to about 7 μM. PMA andrelated molecules, used to increase proliferation, may be used at aconcentration of about 1 μg/ml to 500 μg/ml, preferably at aconcentration of about 10 μg/ml to 200 μg/ml. The glycosaminoglycan,heparan sulfate, is a ubiquitous component on the surface of mammaliancells known to affect a variety of cellular processes, and which bindsto growth factor molecules such as FGF and amphiregulin, therebypromoting the binding of these molecules to their receptors on thesurfaces of cells. It can be added to the culture medium in combinationwith other biological factors, at a concentration of about 1 ng/ml to 1mg/ml; more preferred is a concentration of about 0.2 μg/ml to 20 μg/ml,most preferred is a concentration of about 2 μg/ml.

Using these screening methods, it is possible to screen for potentialdrug side-effects on pre- and post-natal CNS cells by testing for theeffects of the biological agents on stem cell and progenitor cellproliferation and on progenitor cell differentiation or the survival andfunction of differentiated CNS cells. The proliferated precursor cellsare typically plated at a density of about 5×10⁶ cells/ml. If it isdesired to test the effect of the biological agent on a particulardifferentiated cell type or a given make-up of cells, the ratio ofneurons to glial cells obtained after differentiation can be manipulatedby separating the different types of cells. For example, the O4 antibody(available from Boerhinger Mannheim) binds to oligodendrocytes and theirprecursors. Using a panning procedure, oligodendrocytes are separatedout. Astrocytes can be panned out after a binding procedure using theRAN 2 antibody (available from ATCC). Tetanus toxin (available fromBoerhinger Mannheim) can be used to select out neurons. By varying thetrophic factors added to the culture medium used during differentiationit is possible to intentionally alter the phenotype ratios. Such trophicfactors include EGF, FGF, BDNF, CNTF, TGFα, GDNF, and the like. Forexample, FGF increases the ratio of neurons, and CNTF increases theratio of oligodendrocytes. Growing the cultures on beds of glial cellsobtained from different CNS regions will also affect the course ofdifferentiation as described above. The differentiated cultures remainviable (with phenotype intact) for at least a month.

The effects of the biological agents are identified on the basis ofsignificant difference relative to control cultures with respect tocriteria such as the ratios of expressed phenotypes (neurons: glialcells, or neurotransmitters or other markers), cell viability andalterations in gene expression. Physical characteristics of the cellscan be analyzed by observing cell and neurite morphology and growth withmicroscopy. The induction of expression of new or increased levels ofproteins such as enzymes, receptors and other cell surface molecules, orof neurotransmitters, amino acids, neuropeptides and biogenic amines canbe analyzed with any technique known in the art which can identify thealteration of the level of such molecules. These techniques includeimmunohistochemistry using antibodies against such molecules, orbiochemical analysis. Such biochemical analysis includes protein assays,enzymatic assays, receptor binding assays, enzyme-linked immunosorbantassays (ELISA), electrophoretic analysis, analysis with high performanceliquid chromatography (HPLC), Western blots, and radioimmune assays(RIA). Nucleic acid analysis such as Northern blots and PCR can be usedto examine the levels of mRNA coding for these molecules, or for enzymeswhich synthesize these molecules.

The factors involved in the proliferation of stem cells and theproliferation, differentiation and survival of stem cell progeny, and/ortheir responses to biological agents can be isolated by constructingcDNA libraries from stem cells or stem cell progeny at different stagesof their development using techniques known in the art. The librariesfrom cells at one developmental stage are compared with those of cellsat different stages of development to determine the sequence of geneexpression during development and to reveal the effects of variousbiological agents or to reveal new biological agents that alter geneexpression in CNS cells. When the libraries are prepared fromdysfunctional tissue, genetic factors may be identified that play a rolein the cause of dysfunction by comparing the libraries from thedysfunctional tissue with those from normal tissue. This information canbe used in the design of therapies to treat the disorders. Additionally,probes can be identified for use in the diagnosis of various geneticdisorders or for use in identifying neural cells at a particular stagein development.

Electrophysiological analysis can be used to determine the effects ofbiological agents on neuronal characteristics such as resting membranepotential, evoked potentials, direction and ionic nature of current flowand the dynamics of ion channels. These measurements can be made usingany technique known in the art, including extracellular single unitvoltage recording, intracellular voltage recording, voltage clamping andpatch clamping. Voltage sensitive dyes and ion sensitive electrodes mayalso be used.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the scope of the invention, as definedby the appended claims.

EXAMPLE 1 Dissociation of Embryonic Neural Tissue

14-day-old CD₇ albino mouse embryos (Charles River) were decapitated andthe brain and striata were removed using sterile procedure. Tissue wasmechanically dissociated with a fire-polished Pasteur pipette intoserum-free medium composed of a 1:1 mixture of Dulbecco's modifiedEagle's medium (DMEM) and F-12 nutrient (Gibco). Dissociated cells werecentrifuged at 800 r.p.m. for 5 minutes, the supernatant aspirated, andthe cells resuspended in DMEM/F-12 medium for counting.

EXAMPLE 2 Dissociation of Adult Neural Tissue

Brain tissue from juvenile and adult mouse brain tissue was removed anddissected into 500 μm sections and immediately transferred into lowcalcium oxygenated artificial cerebrospinal fluid (low Ca²⁺ aCSF)containing 1.33 mg/ml trypsin, 0.67 mg/ml hyaluronidase, and 0.2 mg/mlkynurenic acid. Tissue was stirred in this solution for 90 minutes at32° C.-35° C. aCSF was poured off and replaced with fresh oxygenatedaCSF for 5 minutes. Tissue was transferred to DMEM/F-12/10% hormonesolution containing 0.7 mg/ml ovomucoid and triturated with a firepolished pasteur pipette. Cells were centrifuged at 400 rpm. for 5minutes, the supernatant aspirated and the pelleted cells resuspended inDMEM/F-12/10% hormone mix.

EXAMPLE 3 Proliferation of Neural Stem Cells on Substrates

2500 cells/cm² prepared as in Example 1 were plated onpoly-L-omithine-coated (15 μg/ml;Siqma) glass coverslips in 24 wellNunclon (0.5 ml/well) culture dishes. The culture medium was aserum-free medium composed of DMEM/F-12 (1:1) including glucose (0.6%),glutamine (2 μM), sodium bicarbonate (3 mM), and HEPES(4-[2hydroxyethyl]-1-piperazineethanesulfonic acid) buffer (5 mM) (allfrom Sigma except glutamine [Gibco]). A defined hormone mix and saltmixture (Sigma) that included insulin (25 μg/ml), transferrin (100μg/ml), progesterone (20 nM), putrescine (60 μM), and selenium chloride(30 nM) was used in place of serum. Cultures contained the above medium,hereinafter referred to as "Complete Medium" together with 16-20 ng/mlEGF (purified from mouse sub-maxillary, Collaborative Research) or TGFα(human recombinant, Gibco). After 10-14 days in vitro, media (DMEM onlyplus hormone mixture) and growth factors were replaced. This mediumchange was repeated every two to four days. The number of survivingcells at 5 days in vitro was determined by incubating the coverslips in0.4% trypan blue (Gibco) for two minutes, washing with phosphatebuffered saline (PBS, pH 7.3) and counting the number of cells thatexcluded dye with a Nikon Diaphot inverted microscope.

EXAMPLE 4 Proliferation of Embryonic Mouse Neural Stem Cells inSuspension

Dissociated mouse brain cells prepared as in Examples 1 and 2 (at 1×10⁵cell/ml) were suspended in Complete Medium with 20 ng/ml of EGF or TGFα.Cells were seeded in a T25 culture flask and housed in an incubator at37° C., 100% humidity, 95% air/5% CO₂. Cells began to proliferate within3-4 days and due to a lack of substrate lifted off the floor of theflask and continued to proliferate in suspension forming clusters ofundifferentiated cells, referred to herein as "neurospheres". After 6-7days in vitro the proliferating clusters (neurospheres) were fed every2-4 days by gentle centrifugation and resuspension in DMEM with theadditives described above.

EXAMPLE 5 Proliferation of Adult Mouse Neural Stem Cells in Suspension

The striata, including the subependymal region, of female, pathogen-freeCD1 albino mice [3 to 18 month old; Charles River (CF1 and CF2 strainsyielded identical results)] were dissected and hand cut with scissorsinto 1-mm coronal sections and transferred into aCSF (pH 7.35, approx.180 mOsmol), aerated with 95% O₂ -5% CO₂ at room temperature. After 15minutes the tissue sections were transferred to a spinner flask (BellcoGlass) with a magnetic stirrer filled with low-Ca²⁺ aCSF (pH 7.35,approx. 180 mOsmol), aerated with 95% O₂ -5% CO₂ at 32 to 35° C.,containing 1.33 mg/ml of trypsin (9000 BAEE units/mg), 0.67 mg/ml ofhyaluronidase (2000 units/mg) and 0.2 mg/ml of kynurenic acid. After 90minutes, tissue sections were transferred to normal aCSF for 5 minutesprior to trituration. Tissue was transferred to DMEM/F-12 (A: 1, Gibco)medium containing 0.7 mg/ml ovomucoid (Sigma) and trituratedmechanically with a fire-narrowed pasteur pipet. Cells were plated (1000viable cells per plate) in noncoated 35 mm culture dishes (Costar)containing Complete Medium and EGF [20 ng/ml, purified from mousesub-maxillary gland (Collaborative Research)] or human recombinant(Gibco/BRL). Cells were allowed to settle for 3-10 minutes after whichthe medium was aspirated away and fresh DMEM/F-12/hormone mix/EGF wasadded. After 5-10 days in vitro the number of spheres (neurospheres)were counted in each 35 mm dish.

EXAMPLE 6 Passaging Proliferated Stem Cells

After 6-7 days in vitro, individual cells in the neurospheres fromExample 4 were separated by triturating the neurospheres with a firepolished pasteur pipette. Single cells from the dissociated neurosphereswere suspended in tissue culture flasks in DMEM/F-12/10% hormone mixtogether with 20 ng/ml of EGF. A percentage of dissociated cells beganto proliferate and formed new neurospheres largely composed ofundifferentiated cells. The flasks were shaken well and neurosphereswere allowed to settle in the bottom comer of the flask. Theneurospheres were then transferred to 50 ml centrifuge tubes andcentrifuged at 300 rpm for 5 minutes. The medium was aspirated off, andthe neurospheres were resuspended in 1 ml of medium containing EGF. Thecells were dissociated with a fire-narrowed pasteur pipette andtriturated forty times. 20 microliters of cells were removed forcounting and added to 20 microliters of Trypan Blue diluted 1:2. Thecells were counted and replated at 50,000 cells/ml. This procedure canbe repeated weekly and results in a logarithmic increase in the numberof viable cells at each passage. The procedure is continued until thedesired number of stem cell progeny is obtained.

EXAMPLE 7 Differentiation of Neural Stem Cell Progeny andImmunocytochemistry

Cells proliferated from Examples 4 and 6 were induced to differentiateby maintaining the cells in the culture flasks in the presence of EGF orTGFα at 20 ng/ml without reinitiating proliferation by dissociation ofthe neurospheres or by plating on poly-ornithine in the continuedpresence of EGF or TGFα.

Indirect immunocytochemistry was carried out with cells prepared as inExample 3 which had been cultured for 14-30 days in vitro on glasscoverslips. For anti-NSE (or anti-nestin) and anti-GFAPimmunocytochemistry, cells were fixed with 4% paraformaldehyde in PBSand 95% ethanol/5% acetic acid, respectively. Following a 30 minutefixation period, coverslips were washed three times (10 minutes each) inPBS (pH=7.3) and then incubated in the primary antiserum (NSE 1:300,nestin 1:1500 or GFAP 1:100) in PBS/10% normal goat serum/0.3%TRITON®-X-100) for two hours at 37 C. Coverslips were washed three times(10 minutes each) in PBS and incubated with secondary antibodies(goat-anti-rabbit-rhodamine for anti-NSE or anti-nestin andgoat-anti-mouse-fluorescein for antiGFAP, both at 1:50) for 30 minutesat 37° C. Coverslips were then washed three times (10 minutes each) inPBS, rinsed with water, placed on glass slides and coverslipped usingFluorsave, a mounting medium preferable for use withfluorescein-conjugated antibodies. Fluorescence was detected andphotographed with a Nikon Optiphot photomicroscope.

Neural stem cell progeny were also differentiated using the followingdifferentiation paradigms. The neurospheres used for each paradigm weregenerated as outlined in Examples 4 and 6. All the neurospheres usedwere passaged at least once prior to their differentiation.

Paradigm 1--Rapid differentiation of neurospheres

Six to 8 days after the first passage, the neurospheres were removed andcentrifuged at 400 r.p.m. The EGF-containing supernatant was removed andthe pellet suspended in EGF-free complete medium containing 1% FBS.Neurospheres (approximately 0.5-1.0×10⁶ cells/well) were plated onpoly-L-ornithine-coated (15 μg/ml) glass coverslips in 24 well Nuclon(1.0 ml/well) culture dishes. After 24 hours in culture, the coverslipswere transferred to 12 well (Costar) culture dishes containing completemedium containing 0.5% FBS. The medium was changed every 4-7 days. Thisdifferentiation procedure is referred to as the "Rapid DifferentiationParadigm" or RDP.

Paradigm 2--Differentiation of dissociated neurospheres

Six to 8 days after the first passage, the neurospheres were removed andcentrifuged at 400 r.p.m. The EGF-containing media was removed and thepellet was suspended in EGF-free complete medium containing 1% FBS. Theneurospheres were mechanically dissociated into single cells with afire-polished Pasteur pipette and centrifuged at 800 r.p.m. for 5minutes. Between 0.5×10⁶ and 1.0×10⁶ cells were plated onpoly-L-ornithine-coated (15 μg/ml) glass coverslips in 24 well Nuclon(1.0 ml/well) culture dishes. The EGF-free culture medium containing 1%FBS was changed every 4-7 days.

Paradigm 3--Differentiation of single neurospheres

Neurospheres were washed free of EGF by serial transfers through changesof EGF-free medium. A single neurosphere was plated ontopoly-L-ornithine-coated (15 μg/ml) glass coverslips in a 24-well plate.The culture medium used was complete medium with or without 1% FBS. Themedium was changed every 4-7 days.

Paradigm 4--Differentiation of single dissociated neurospheres

Neurospheres were washed free of EGF by serial transfers through changesof EGF-free medium. A single neurosphere was mechanically dissociated ina 0.5 ml Eppendorf centrifuge tube and all the cells were plated onto a35 mm culture dish. Complete medium was used with or without 1% FBS.

Paradigm 5--Differentiation of neurospheres co-cultured with striatalastrocytes

Neurospheres, derived from striatal cells as described in Example 1 werelabeled with 5-bromodeoxyuridine (BrdU) and washed free of EGF. Anastrocyte feeder layer was generated from striatal tissue of postnatalmice (0-24 hours), and plated on poly-L-ornithine-coated glasscoverslips in a 24-well culture dish. When the astrocytes wereconfluent, a dissociated or intact neurosphere was placed on eachastrocyte bed. Complete medium was changed after the first 24 hours andthen every forty-eight hours. When differentiated on an astrocyte feederlayer, in addition to GABAergic and substance P-ergic neurons,somatostatin, NPY, glutamate and methenkephalin-containing neurons werepresent.

EXAMPLE 8 Effect of Growth Factors on Neurosphere Differentiation

The effects of CNTF, FGF-2, BDNF, and Retinoic Acid on neurospheredifferentiation were tested using the differentiation paradigms setforth in Example 7.

CNTF

The effect of CNTF was assayed in paradigms 1 and 3. For both paradigms,CNTF was added either at the beginning of the experiment at aconcentration of 10 ng/ml or daily at a concentration of 1 ng/ml. Inparadigm 1, the addition of CNTF increased the number ofNSE-immunoreactive cells in addition to the number oftau-1-immunoreactive cells, suggesting that CNTF has an effect on theproliferation, survival, or differentiation of neurons. Preliminarytesting with antibodies recognizing the neurotransmitters GABA andsubstance P suggest that there is no increase in the number of cellscontaining these proteins. This suggests that a different neuronalphenotype is being produced.

Three different antibodies directed against 04, galactocerebroside(GalC) and MBP were used to study the effect of CNTF on theoligodendrocytes of paradigm 1. CNTF had no effect on the number ofO4(+) cells, but there was an increase in the number of GalC(+) andMBP(+) cells compared with the control. Thus it appears that CNTF playsa role in the maturation of oligodendrocytes.

In one experiment, the neurospheres were differentiated as outlined inparadigm 1 except that serum was never added to the culture medium.While the effect of CNTF on neurons and oligodendrocytes was not asapparent as in the presence of serum, there was an increase in theproliferation of flat, protoplasmic astrocytes. Hence, CNTF will affectastrocyte differentiation in various culture conditions.

In paradigm 3, the addition of CNTF resulted in an increase in thenumber of NSE(+) cells.

BDNF

The effect of BDNF was tested using Paradigm 3. There was an increase inthe number of NSE(+) neurons per neurosphere. Additionally, there was anincrease in the neuronal branching and the migration of the neurons awayfrom the sphere.

FGF-2

The effect of FGF-2 was tested using paradigms 2 and 4. In paradigm 2,20 ng/ml of FGF-2 was added at the beginning of the experiment and cellswere stained 7 days later. FGF-2 increased the number of GFAP(+) cellsand the number of NSE(+) cells. This suggests that FGF-2 has aproliferative or survival effect on the neurons and astrocytes.

In paradigm 4, 20 ng/ml of FGF-2 was added at the beginning of theexperiment and assayed 7-10 days later. FGF-2 induced the proliferationof neural stem cell progeny generated by the EGF-responsive stem cell.It induced two different cell types to divide, neuroblasts andbipotential progenitor cells. The neuroblast produced, on average, 6neurons while the bipotential cell produced approximately 6 neurons anda number of astrocytes.

In previous studies, it was found that when plated at low density (2500cells/cm²), addition of EGF up to 7 days in vitro (DIV) could initiateproliferation of the stem cell, but not if applied after 7 DIV. Striatalcells (E14, 2500 cell/cm²) were plated in the absence or presence of 20ng/ml of FGF-2. After 11 DIV, cultures were washed and medium containing20 ng/ml of EGF was added. After 4-5 DIV, in cultures that were primedwith FGF-2, greater than 70% of the wells examined contained clusters ofproliferating cells that developed into colonies with the morphologicand antigenic properties of the EGF-generated cells. Cultures that hadnot been primed with FGF-2 showed no EGF-responsive proliferation. Thesefindings suggest that the EGF-responsive stem cells possess FGF-2receptors that regulate its long term survival.

Retinoic acid

The effect of retinoic acid at 10⁻⁷ M was tested using paradigm 1. Therewas an increase in the number of NSE(+) and tau-1(+) cells, suggestingthat retinoic acid increases the number of neurons.

EXAMPLE 9 Proliferation of Embryonic Human Neural Stem Cells andDifferentiation of the Neural Stem Cell Progeny

With approval of the Research Ethical Committee at the University ofLund and the Ethics Committee at the University of Calgary, nine 8-12week old human fetuses were obtained by suction abortions. Tissue wasdissected and any identifiable brain regions were removed. Within 4-5days post-dissection, tissue pieces were mechanically dissociated intosingle cells using the procedure of Example 1 and the number of viablecells was counted. About 0.1×10⁶⁻⁰.5×10⁶ cells were plated in 35 cm²tissue culture flasks (without substrate pretreatment) in CompleteMedium with 20 ng/ml of human recombinant EGF (Gibco/BRL).

Two to three days after plating the cells, the majority of the viablecells had extended processes and had taken on a neuronal morphology. Byseven days in vitro (DIV), the neuronal-like cells began to die and by14 DIV nearly all of these cells were dead or dying (determined by theabsence of processes, irregular membranes and granular cytoplasm). A fewof the cells (1%) did not extend any processes or flatten nor did theytake on an astrocytic morphology, instead these cells remained roundedand by 5 to 7 DIV began to divide. By 10 to 14 DIV, small clusters ofcells, attached to the substrate, were identified. During the next 7 to10 days (17 to 24 DIV), these small clusters continued to grow in sizeand many remained attached to the substrate. By 28 to 30 DIV, nearly allthe proliferating clusters had lifted off the substrate and werefloating in suspension. While floating in suspension, the clusterscontinued to grow in size and were passaged after they had been inculture for 30 to 40 days using the procedure described in Example 6.EGF-responsive cells began to proliferate after a few DIV and formedfloating spheres that were passaged a second time after 30 to 40 DIV.

Thirty to 60 days after passage two or three, 2-3 ml aliquots containingmedia and pass 2 spheres were taken from the tissue culture flasks andplated onto 35 mm culture dish. Single spheres were placed ontopoly-L-ornithine coated glass coverslips in DMEM/F-12/HM mediumcontaining EGF. Spheres immediately attached to the substrate and withinthe first 24×48 hours cells begin to migrate from the sphere. At 14 DIVcells continued to proliferate and migrate resulting in an increase inthe diameter of the transferred sphere. By 30 DIV, a large number ofcells had been generated from the original sphere and had migrated at asimilar rate from the center producing a concentric circle of associatedcells. At the periphery, the majority of the cells were one cell layerthick while closer to the center there were denser regions of cells.

Forebrain regions from eight week old tissue produced no spheres, whilespheres were observed from hindbrain tissue in two of the four eightweek old samples. For the nine week old fetuses, spheres were generatedfrom forebrain region in two of the four samples and in two of the threehindbrain regions which were received. The twelve week old fetuscontained only hindbrain tissue and spheres were produced.

Spheres generated from primary culture or pass 1 spheres were removedfrom the tissue culture flask, without inducing differentiation, andplated onto poly-L-ornithine coated glass coverslips in DMEM/F-12/HMmedium for two hours to allow the spheres to attach to the substrate.Coverslips were removed and processed for indirect immunohistochemistry.Immunostaining with antibodies directed against neurofilaments (168 kDa)or GFAP did not identify any immunoreactive (IR) cells. However, nearlyall of the cells were immunoreactive with an antibody that recognizeshuman nestin.

Thirty to 45 days after being plated onto the poly-L-omithine coatedsubstrate, cells were fixed and processed for indirectimmunocytochemical analysis with antibodies directed against: MAP-2,Tau-1, neurofilament 168 kDA, GABA, substance P (neuronal markers); GFAP(astrocyte marker); 04 and MBP (oligodendrocyte markers). Numerous MAP-2and Tau-1-IR cell bodies and processes were identified in addition to alarge number of Tau-1-IR fibers. While there was no indication ofsubstance P immunoreactivity, GABA-IR cell bodies with long branchedprocesses were seen. Neurofilament-IR cells were strongly IR for GFAP.O4-IR cells with an O-2A morphology and an oligodendrocyte morphologywere present. MBP-IR (found on oligodendrocytes) was also seenthroughout the cultures.

EXAMPLE 10 Proliferation of Adult Monkey (Rhesus) Neural Stem Cells andDifferentiation of the Neural Stem Cell Progeny

The conus medularis was removed from an adult male monkey (Rhesus) andhand cut with scissors into 1-mm sections and transferred intoartificial cerebrospinal fluid (aCSF) containing 124 mM NaCl, 5 mM KCl,1.3 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃, and 10 mM D-glucose (pH 7.35,approx. 280 mOsmol), aerated with 95% O₂ -5% CO₂ at room temperature.After 15 min, the tissue sections were transferred to a spinner flask(Bellco Glass) with a magnetic stirrer filled with low-Ca²⁺ aCSFcontaining 124 mM NaCl, 5 mM KCl, 3.2 mM MgCl₂, 0.1 mM CaCl₂, 26 mMNaHCO₃, and 10 mM D-glucose (pH 7.35, approx. 280 mOsmol), aerated with95% O₂ -5% CO₂ at 32 to 35° C., containing 1.33 mg/ml of trypsin (9000BAEE units/mg), 0.67 mg/ml of hyaluronidase (2000 units/mg) and 0.2mg/ml of kynurenic acid. After 90 min, tissue sections were transferredto normal aCSF for 5 min prior to trituration. Tissue was transferred toDMEM/F-12 (1:1, Gibco) medium containing 0.7 mg/ml ovomucoid (Sigma) andtriturated mechanically with a fire-narrowed pasteur pipet.

Cells were plated (1000 viable cells per plate) in non-coated 35 mmculture dishes (Costar) containing Complete Medium and 20 ng/ml EGF(human recombinant from Gibco/BRL). After 7 to 10 days in culture,floating spheres were transferred with wide-bore pipets onto laminin (15μg/ml)(Sigma)-coated glass coverslips in 24-well culture dishes. EGF @20 ng/ml was added to the medium. Spheres attached to the substrate andcells within the sphere continued to proliferate. After 14 to 21 days invitro (DIV), the cells were probed by indirect immunocytochemistry forthe presence of neuron, astrocytes and oligodendrocytes. All three celltypes were identified.

EXAMPLE 11 Proliferation of Adult Human Neural Stem Cells andDifferentiation of the Neural Stem Cell Progeny

During a routine biopsy, normal tissue was obtained from a 65 year oldfemale patient. The biopsy site was the right frontal lobe, 6 mm fromthe tip of the frontal/anterior horn of the lateral ventricle. Thetissue was dissociated using the procedure outlined in Example 2 andcultured in Complete Medium with EGF and FGF-2 (20 ng/ml of each growthhormone), in T25 flasks (Nunclon). The flasks were examined every 2-3days for neurosphere formation. Clonally-derived cells were passagedusing single sphere dissociation: single neurospheres were triturated100× in sterile aliquot tubes containing 200 μl of themedia/hormone/EGF-FGF-2 solution before culturing in 24- or 96-wellplates. First-passage neurospheres were plated on poly-ornithine andlaminin coated coverslips and allowed to plate down for 14 days inmedia/hormone/EGF + FGF-2. Some first passage neurospheres were platedon laminin (20 μg/ml) and poly-ornithine coated coverslips inmedia/hormone mix for 19 hours, then processed for nestin staining asoutlined in Example 7. Nestin staining indicated that the neurospheres,prior to the induction of differentiation (as described below) werenestin positive, indicative of the presence of immature undifferentiatedcells.

Pass one human neurospheres were plated on a laminin coated substrate(see above). After 14 days, the cultures received a media change tomedia/hormone mix plus 1% FBS and were allowed to differentiate for 7days. Immunocytochemical analysis was then performed to determinedifferent neural phenotypes. The differentiated cells were fixed with 4%paraformaldehyde in PBS for 20 minutes. The coverslips were washed threetimes (five minutes each) in PBS. For triple label immunocytochemistry,the cells were permeabilized for 5 minutes in .3% Triton-X in PBSfollowed by 2 washes with PBS. A first set of primary antibodies, MAP-2(mouse monoclonal, 1:1000, Boerhinger Mannheim) and GFAP (Rabbitpolyclonal, 1:300, BTI), used to determine the presence of neurons andastrocytes respectively, were mixed in 10% normal goat serum in PBS. Thecells were incubated at 37° C. for 2 hours and then washed 3 times inPBS. A first set of secondary antibodies, goat anti-mouse rhodamine(Jackson Immuno Research) and goat anti-rabbit FITC (IgG, 1:100 JacksonImmuno Research) were mixed in PBS. The cells were incubated for 30minutes at 37° C. and then rinsed three times with PBS. The secondprimary antibody, O4 (mouse monoclonal IgM, 1:10) for oligodendrocytes,was mixed in 10% normal goat serum in PBS. The cells were incubated for2 hours at 37° C. The second set of secondary goat anti-mouse AMCA IgM(1:100 Jackson Immuno Research) was mixed in PBS and cells wereincubated for 30 minutes at 37° C. The cells were then rinsed twice inPBS and then in double distilled water before mounting with Fluorosave.

EXAMPLE 12 Screening for the trkB Receptor on Neural Stem Cell Progeny

The expression of the trk family of neurotrophin receptors inEGF-generated neurospheres was examined by northern blot analysis. TotalmRNA was isolated from mouse and rat striatal EGF-generatedneurospheres. Both rat and mouse neurospheres expressed high levels oftrkB receptor mRNA, but did not express trk nor trkC mRNA. Inpreliminary experiments, single EGF-generated mouse neurospheres wereplated on poly-L-ornithine coated glass coverslips and cultured in theabsence or presence of 10 ng/ml of BDNF. When examined after 14-28 daysin vitro, neurospheres plated in the presence of BDNF contained NSE(+)cells with extensive and highly branched processes; well-developedNSE(+) cells were not observed in the absence of BDNF. Activation of thetrkB receptor on EGF-generated neurospheres may enhance differentiation,survival of and/or neurite outgrowth from newly generated neurons.

EXAMPLE 13 Screening for the GAP43 Membrane Phosphoprotein on NeuralStem Cell Progeny

Growth-associated protein (GAP-43) is a nervous system-specific membranephosphoprotein which is down-regulated during development. Originally,GAP-43 was though to be neuron-specific, however, recent reportsindicate that this protein may be at least transiently expressed duringdevelopment in some astrocytes, oligodendrocytes and in Schwann cells.At present, the role of GAP-43 in macroglia is not known. The transientexpression of GAP-43 in glial cells generated from the EGF-responsivestem cells derived from embryonic and adult murine striatum wasinvestigated. Glial cell (astrocyte and oligodendrocyte) differentiationwas induced by plating neural stem cell progeny in a medium containing1% FBS with no EGF. The cells were then probed with specific antibodiesfor GAP-43, nestin, GFAP, O4, and GalC. In order to identify cellsexpressing GAP-43, the antibodies were pooled in various combinationsusing dual-label immunofluorescence methods.

During the first two days post plating, there was a low to moderatelevel of GAP43 expression in almost all cells (flat, bipolar andstellate), but by 3-4 days postplating, the level of GAP-43 expressionbecame restricted to the bipolar and stellate cells. At 4 days themajority of GAP-43-expressing cells co-labelled with the oligodendrocytemarkers O4 and GalC although GFAP and GAP-43 was coexpressed in a numberof cells. At one week post-plating however, essentially all of theGFAP-expressing astrocytes no longer expressed GAP-43 while the majorityof the O4 and GalC-expressing cells continued to express GAP-43. At 7-10days, these oligodendrocytes began to express MBP and lose theexpression of GAP-43. The EGF-responsive stem cells may represent auseful model system for the study of the role of GAP-43 in glial andneuronal development.

EXAMPLE 14 Treatment of Neurodegenerative Disease Using Progeny of HumanNeural Stem Cells Prolifetated In Vitro

Cells are obtained from ventral mesencephalic tissue from a human fetusaged 8 weeks following routine suction abortion which is collected intoa sterile collection apparatus. A 2×4×1 mm piece of tissue is dissectedand dissociated as in Example 1. Neural stem cells are then proliferatedas in Example 4. Neural stem cell progeny are used forneurotransplantation into a blood-group matched host with aneurodegenerative disease. Surgery is performed using a BRW computedtomographic (CT) stereotaxic guide. The patient is given localanesthesia supplemencea with intravenously administered midazolam. Thepatient undergoes CT scanning to establish the coordinates of the regionto receive the transplant. The injection cannula consists of a 17-gaugestainless steel outer cannula with a 19-gauge inner stylet. This isinserted into the brain to the correct coordinates, then removed andreplaced with a 19-gauge infusion cannula that has been preloaded with30 μl of tissue suspension. The cells are slowly infused at a rate of 3μl/min as the cannula is withdrawn. Multiple stereotactic needle passesare made throughout the area of interest, approximately 4 mm apart. Thepatient is examined by CT scan postoperatively for hemorrhage or edema.Neurological evaluations are performed at various post-operativeintervals, as well as PET scans to determine metabolic activity of theimplanted cells.

EXAMPLE 15 Remyelination of Myelin Deficient Rats Using Neural Stem CellProgeny Proliferated In Vitro

Embryonic day 15 (E15) Sprague Dawley rats and E14-15 mice were obtainedand the neural tissue was prepared using the methods described inExample 1. The cells were suspended in Complete Medium with 16-20 ng/mlEGF (purified from mouse submaxillary, Collaborative Research) or TGFα(human recombinant, Gibco). The cells were seeded in a T25 culture flaskand housed in an incubator at 37° C., 100% humidity, 95% air/5% CO₂ andproliferated using the suspension culture method of Example 4. Cellsproliferated within 3-4 days and, due to lack of substrate, lifted offthe floor of the flask and continued to proliferate in suspensionforming neurospheres.

After 6-8 days in vitro (DIV) the neurospheres were removed, centrifugedat 400 r.p.m. for 2-5 minutes, and the pellet mechanically dissociatedinto individual cells with a fire-polished glass pasteur pipet. Cellswere replated in the growth medium where proliferation of the stem cellsand formation of new neurospheres was reinitiated.

Litters of first day postnatal myelin deficient rats were anesthetizedusing ice to produce hypothermia. Myelin deficiency is an X-linked traitand thus only one half of the males in any litter are affected.Therefore, only the males were used for these studies. Onceanesthetized, a small rostral to caudal incision was made at the levelof the lumbar enlargement. The muscle and connective tissue was removedto expose the vertebral laminae. Using a fine rat tooth forceps, onelamina at the lumbar enlargement was removed and a small cut is made inthe dura mater to expose the spinal cord.

A stereotaxic device holding a glass pipet was used to inject a 1 μlaliquot of the cell suspension (approximately 50,000 cells/μl) describedabove. The suspension is slowly injected into a single site (althoughmore could be done) in the dorsal columns of the spinal cord. Ascontrols, some of the animals were sham-injected with sterile saline.The animals were marked by clipping either toes or ears to distinguishbetween both experimental groups. Following injection of the cellsuspension, the wound was closed using sutures or stainless steel woundclips and the animals were revived by warming on a surgical heating padand then returned to their mother.

The animals were allowed to survive for two weeks post-injection andwere then deeply anesthetized with nembutal (150 mg/kg) and perfusedthrough the left ventricle. The spinal cords were removed and the tissueexamined by light and electron microscopy. Patches of myelin were foundin the dorsal columns of the recipients of both rat and mouse cells,indicating that neural stem cells isolated from rat and mouse neuraltissue can differentiate into oligodendroglia and are capable ofmyelination in vivo.

Because the myelin deficient rat spinal cord is almost completely devoidof myelin, myelin formed at or near the site of injection is derivedfrom the implanted cells. It is possible that the process of injectionwill allow for the entry of Schwann cells (myelinating cells of the PNS)into the spinal cord. These cells are capable of forming myelin withinthe CNS but can be easily distinguished from oligodendrocytes usingeither light microscopy or immunocytochemistry for CNS myelin elements.There is usually a very small amount of CNS myelin within the myelindeficient rat spinal cord. This myelin can be distinguished from normaldonor myelin based on the mutation within the gene for the major CNSmyelin protein, proteolipid protein (PLP). The myelin deficient ratmyelin is not immunoreactive for PLP while the donor myelin is.

EXAMPLE 16 Remyelination in Human Neuromyelitis Optica

Neuromyelitis optica is a condition involving demyelination ofprincipally the spinal cord and optic nerve. Onset is usually acute andin 50% of the cases death occurs within months. The severity ofdemyelination as well as lesion sites can be confirmed by magneticresonance imaging (MRI).

Neural stem cell progeny are prepared from fetal human tissue by themethods of Example 9 or 14. Cells are stereotactically injected into thewhite matter of the spinal cord in the vicinity of plaques as visualizedby MRI. Cells are also injected around the optic nerve as necessary.Booster injections may be performed as required.

EXAMPLE 17 Remyelination in Human Pelizaeus-Merzbacher Disease

Pelizaeus-Merzbacher disease is a condition involving demyelination ofthe CNS. The severity of demyelination as well as lesion sites can beconfirmed by magnetic resonance imaging (MRI).

Neural stem cell progeny are prepared from fetal human tissue by themethods of Examples 9 or 14. Cells are stereotactically injected intothe white matter of the spinal cord in the vicinity of plaques asvisualized by MRI. Cells are also injected around the optic nerve asnecessary. Booster injections may be performed as required.

EXAMPLE 18 Genetic Modification of Neural Stem Cell Progeny

Cells proliferated as in Examples 3 or 4 are transfected with expressionvectors containing the genes for the FGF-2 receptor or the NGF receptor.Vector DNA containing the genes are diluted in 0.1× TE (1 mM Tris pH8.0, 0.1 mM EDTA) to a concentration of 40 μg/ml. 22 μl of the DNA isadded to 250 μl of 2×HBS (280 mM NaCl, 10 mM KCl, 1.5 mM Na₂ HPO₄.2H₂ O,12 mM dextrose, 50 mM HEPES) in a disposable, sterile 5 ml plastic tube.31 μl of 2 M CaCl₂ is added slowly and the mixture is incubated for 30minutes at room temperature. During this 30 minute incubation, the cellsare centrifuged at 800 g for 5 minutes at 4° C. The cells areresuspended in 20 volumes of ice-cold PBS and divided into aliquots of1×10⁷ cells, which are again centrifuged. Each aliquot of cells isresuspended in 1 ml of the DNA-CaCl₂ suspension, and incubated for 20minutes at room temperature. The cells are then diluted in growth mediumand incubated for 6-24 hours at 37° C. in 5%-7% CO². The cells are againcentrifuged, washed in PBS and returned to 10 ml of growth medium for 48hours.

The transfected neural stem cell progeny are transplanted into a humanpatient using the procedure described in Example 14, or are used fordrug screening procedures as described in the examples below.

EXAMPLE 19 Genetic Modification of Neural Stem Cell Progeny With aRetrovirus Containing the Bacterial B-Galactosidase Gene

Neural stem cell progeny were propagated as described in Example 4. Alarge pass-1 flask of neurospheres (4-5 days old) was shaken to dislodgethe spheres from the flask. The flask was spun at 400 r.p.m. for 3-5minutes. About half of the media was removed without disturbing theneurospheres. The spheres and the remaining media were removed, placedinto a Falcon 12 ml centrifuge tube, and spun at 600 r.p.m. for 3-5minutes. The remaining medium was removed, leaving a few hundredmicroliters.

A retrovirus which contained the bacterial B-galactosidase gene waspackaged and secreted, in a replication-deficient fashion, by the CREBAG2 cell line produced by C. Cepko. A day after the CRE cells reachedconfluence, the cells were washed with PBS and the retrovirus wascollected in DMEM/F12/HM/20 ng/ml EGF for four days. Thevirus-containing media was filtered through a 0.45 μm syringe filter.The neurospheres were resuspended in the virus-containing media,transferred to a large flask, and left in an incubator overnight at 37°C. The next day, the contents of the flask were transferred to a 12 mlcentrifuge tube and spun at 800 r.p.m. The cells were resuspended inEGF-containing media/HM, dissociated into single cells, and counted. Thecells were replated in a large flask at 50,000 cells/ml in a total of 20mls.

Four days later, transformed cells were selected with G418 at aconcentration of 300 μg/ml. Transformed spheres were plated on apoly-ornithine coated glass coverslip in a 24-well plate. After theneurospheres adhered to the plate, the cells were fixed with 0.1%glutaraldehyde for 5 minutes at 4° C. After the cells were fixed, theywere washed twice with PBS for 10 minutes. The cells were then washedwith 0.1% TRITON® in PBS for 10-15 minutes at room temperature. A 1mg/ml X-Gal solution was added to each well and incubated overnight at37° C. After incubation overnight, the cells were washed three timeswith PBS for 10 minutes each and observed for any reaction products. Apositive reaction resulted in a blue color, indicating cells containingthe transferred gene.

EXAMPLE 20 Proliferation of Neural Stem Cells from Transgenic Mice

Transgenic mice were produced using standard pronuclear injection of theMBP-lacZ chimeric gene, in which the promoter for MBP directs theexpression of E. coli B-galactosidase (lacZ). Transgenic animals wereidentified by PCR using oligonucleotides specific for lacZ.

Neurospheres were prepared from E15 transgenic mice and DNA negativelittermates using the procedures set forth in Example 4. Theneurospheres were propagated in the defined culture medium in thepresence of 20 ng/ml EGF and were passaged weekly for 35 weeks. Forpassaging, the neurospheres were harvested, gently centrifuged at 800RPM, and mechanically dissociated by trituration with a fire-polishedPasteur pipet. At various passages, the cells were induced todifferentiate into oligodendrocytes, astrocytes, and neurons by alteringthe culture conditions. The free-floating stem cell clusters were gentlycentrifuged, resuspended in the same base defined medium without EGF andwith 1% FBS and plated on poly ornithine-treated glass coverslips topromote cell attachment. The clusters attach firmly to the glass, andthe cells slow or stop dividing and begin to differentiate.

The identification of various cell types was accomplished usingimmunofluorescence microscopy with antibodies specific for neurons(MAP-2, NFL, and NF-M), astrocytes (GFAP) and oligodendrocytes andoligodendrocyte precursors (A2B5, O₁, O₄, Gal C, and MBP). One to 14days post-plating, the cells on the coverslips were incubated unfixed,for 30 minutes at room temperature with the primary antibodies O1, O4,GalC, and A2B5 (supernatants) diluted in minimal essential medium with5% normal goat serum and 25 mM HEPES buffer, pH 7.3 (MEM-HEPES, NGS).Following the primary antibodies, the coverslips were gently washed 5times in rhodamine-conjugated secondary antibodies (Sigma) diluted inMEM-HEPES, NGS. The coverslips were then washed 5 times in MEM-HEPES andfixed with acid alcohol (5% glacial acetic acid/95% ethanol) at 20° C.The coverslips were then washed 5 times with MEM-HEPES, and eithermounted and examined using fluorescence microscopy or immmunoreactedwith rabbit polyclonal antisera raised against GFAP, nestin, MBP, orproteolipid protein (PLP). When subjected to a second round ofimmunolabeling, the coverslips were incubated first for 1 hour with 5%normal goat serum (NGS) in 0.1 M phosphate buffer with 0.9% NaCl at pH7.4 (PBS) and then incubated in rabbit primary antibodies diluted in NGSfor 1-2 hours at room temperature. The coverslips were washed 3 timeswith PBS and then incubated with the appropriate secondary antibodyconjugates diluted in NGS, washed again with PBS and then finallymounted on glass microscope slides with Citifluor antifadent mountingmedium and examined using a fluorescence microscope. In cases were theywere not incubated first with the monoclonal antibody supernatants, thecoverslips were fixed for 20 minutes with 4% paraformaldehyde in PBS (pH7.4), washed with PBS, permeabilized with 100% ethanol, washed againwith PBS and incubated with 5% NGS in PBS for 1 hour. The primaryantibodies and secondary antibody conjugates were applied as outlinedabove.

The neural stem cells derived from the transgenic animals wereindistinguishable from non transgenic stem cells in their potential fordifferentiation into neurons, astrocytes, and oligodendrocytes. The MBPpromoter directed the expression of the B-galactosidase reporter gene ina cell-specific and developmentally appropriate fashion. The transgeneexpression is highly stable as oligodendrocytes derived from latepassage MBP-lacZ neurospheres (20 passages), expressed theB-galactosidase gene. Thus, transgenically marked neurospheres arelikely to be an excellent source of cells for glial celltransplantation.

EXAMPLE 21 Genetic Modification of Neural Stem Cell Progeny UsingCalcium Phosphate Transfection

Neural stem cell progeny are propagated as described in Example 4. Thecells are then infected using a calcium phosphate transfectiontechnique. For standard calcium phosphate transfection, the cells aremechanically dissociated into a single cell suspension and plated ontissue culture-treated dishes at 50% confluence (50,000-75,000cells/cm²) and allowed to attach overnight. The modified calciumphosphate transfection procedure is performed as follows: DNA (15-25 μg)in sterile TE buffer (10 mM Tris, 0.25 mM EDTA, pH 7.5) diluted to 440μl with TE, and 60 μl of 2 M CaCl₂ (pH to 5.8 with 1 M HEPES buffer) isadded to the DNA/TE buffer. A total of 500 μl of 2× HeBS (HEPES-Bufferedsaline; 275 mM NaCl, 10 mM KCl, 1.4 mM Na₂ HPO₄, 12 mM dextrose, 40 mMHEPES buffer powder, pH 6.92) is added dropwise to this mix. The mixtureis allowed to stand at room temperature for 20 minutes. The cells arewashed briefly with 1× HeBS and 1 ml of the calcium phosphateprecipitated DNA solution is added to each plate, and the cells areincubated at 37° for 20 minutes. Following this incubation, 10 mls ofcomplete medium is added to the cells, and the plates are placed in anincubator (37° C., 9.5% CO₂) for an additional 3-6 hours. The DNA andthe medium are removed by aspiration at the end of the incubationperiod, and the cells are washed 3 times with complete growth medium andthen returned to the incubator.

EXAMPLE 22 Genetically Modified Neural Stem Cell Progeny Expressing NGF

Using either the recombinant retrovirus or direct DNA transfectiontechnique, a chimeric gene composed of the human CMV promoter directingthe expression of the rat NGF gene is introduced into the neurospherecells. In addition, the vector includes the E. coli neomycin resistancegene driven off of a viral promoter. After 2 weeks of G418 selection,the cells are cloned using limiting dilution in 96-multi-well plates andthe resulting clones are assayed for neurotrophin protein expressionusing a neurotrophin receptor (trk family) autophosphorylation bioassay.

Clones expressing high levels of NGF are expanded in T-flasks prior todifferentiation. The cells are then removed from the EGF-containingcomplete medium and treated with a combination of serum and a cocktailof growth factors to induce astrocyte differentiation. The astrocytesare again assayed for NGF expression to ensure that the differentiatedcells continue to express the trophic factors. Astrocytes that secreteNGF are then injected into fimbria/fornix lesioned rat brainsimmediately post-lesioning in order to protect the cholinergic neurons.Control astrocytes that do not secrete NGF are injected into similarlylesioned animals. The sparing of cholinergic neurons in the lesion modelis assessed using immunocytochemistry for ChAT, the marker for thesecholinergic neurons.

EXAMPLE 23 Genetically Modified Neural Stem Cell Progeny Expressing CGAT

Recently, a novel chromaffin granule amine transporter (CGAT) cDNA hasbeen described by Liu et al. (Cell, 70:539-551 (1992)), which affordsresistance to the neurotoxin MPP+ in Chinese hamster ovary (CHO) cellsin vitro. Because dopaminergic neurons from the substantia nigra arespecifically killed by MPP+, CGAT gene expression in geneticallymodified neural stem cell progeny may improve viability of the cellsafter they are implanted into the Parkinsonian brain. Neural stem cellprogeny are propagated as in Example 4. The cells are mechanicallydissociated and plated on plastic dishes and infected with a retroviruscontaining the CGAT cDNA. The expression of the CGAT cDNA (Liu et al.supra) is directed by a constitutive promoter (CMV or SV40, or aretroviral LTR) or a cell-specific promoter (TH or other dopaminergic orcatecholaminergic cell-specific regulatory element or the like). Thecells are screened for the expression of the CGAT protein. Selectedcells can then be differentiated in vitro using a growth factor or acombination of growth factors to produce dopaminergic orpre-dopaminergic neurons.

EXAMPLE 24 3H-Thymidine Kill Studies Identify Presence of ConstitutivelyProliferating Population of Neural Cells in Subependymal Region

Adult male CD1 mice received a series of intraperitoneal injections of3H-thymidine (0.8 ml per injection, specific activity 45-55 mCi/mmole,ICN Biomedical) on day 0 (3 injections, 1 every 4 hours) in order tokill the constitutively proliferating subependymal cells. On day 0.5, 1,2, 4, 6, 8 or 12, animals received 2 BrdU injections 1 hour apart (seeExample 25) and were sacrificed 0.5 hour after the last injection.

It was observed that 10% of the cells were proliferating on day 1post-kill, and by 8 days the number of proliferating cells had reached85%, which was not statistically significantly different from controlvalues. Animals were sacrificed and the brains were removed andprocessed as described in Example 10.

In a second group of animals, 3H-thymidine injections were given on day0 (3 injections, 1 every 4 hours), followed by an identical series ofinjections on day 2 or 4. Animals were allowed to survive for 8 daysfollowing the second series of injections (days 9, 10 and 12respectively) at which time they received 2 injections of BrdU and weresacrificed 0.5 hours later. Animals were sacrificed and the brains wereremoved and processed as described in Example 25.

After the second series of injections on day 2 only 45% of theproliferating population had returned relative to control values. Thisindicates that the second series of injections given on day 2 had killedthe stem cells as they were recruited to the proliferating mode. Thesecond series of injections given on day 4 resulted in a return tocontrol values by day 8 suggesting that by this time, the stem cellswere no longer proliferating and hence were not killed by the day 4series of injections.

EXAMPLE 25 BrdU Labeling Studies Identify Presence of Constitutively

Proliferating Population of Neural Cells in Subependymal Region Adultmale CD1 mice (25-30 g, Charles River) were injected intraperitoneally(i.p.) with bromodeoxyuridine (BrdU, Sigma, 65 mg/kg) every 2 hours fora total of 5 injections in order to label all of the constitutivelyproliferating cells in the subependyma lining the lateral ventricles inthe forebrain. One month later, animals were sacrificed with an overdoseof sodium pentobarbital and transcardially perfused using 4%paraformaldehyde. The brains were removed and post-fixed overnight in 4%paraformaldehyde with 20% sucrose. Brain sections were cut on a cryostat(30 um) and collected in a washing buffer [0.1 M phosphate bufferedsaline (PBS) pH 7.2 with 1% normal horse serum and 0.3% TRITON® X-100].Sections were incubated in 1M HCl at 60° C. for 0.5 hours then washed 3times (10 minutes each) in washing buffer. Following the final wash,sections were incubated in anti-BrdU (Becton Dickinson, 1:25) for 45hours at 4° C. After incubation in the primary antibody, sections werewashed 3 times and incubated for 1 hours in biotinylatedhorse-anti-mouse secondary antibody (Dimension Lab, 1:50) at roomtemperature followed by another 3 washes. The sections were thenincubated for 1 hour in avidin conjugated FITC (Dimension Lab, 1:50) atroom temperature and washed a final 3 times. Sections were mounted ongelatin coated slides, air-dried and coverslipped with Fluoromount.Slides were examined for BrdU positive cells using a NIKON fluorescentmicroscope. The number of BrdU positive cells was counted within thesubependyma surrounding the lateral ventricles in 8 samples in sectionsbetween the closing of the corpus callosum rostrally and the crossing ofthe anterior commissure caudally. It was found that 31 days followingthe series of BrdU injections, 3% of the subependymal cells were stilllabeled compared to control animals sacrificed immediately following theseries of injections (control 100%).

EXAMPLE 26 3H-Thymidine Kill Studies Identify Presence of RelativelyQuiescent Neural Stem Cells in Subependymal Region

Adult male CD1 mice were divided into 4 groups. Group A animals receiveda series of 3H-thymidine injections on day 0 (3 injections, 1 every 4hours). Animals in groups B and C received a series of 3H-thymidineinjections on day 0 followed by a second series of injections on day 2or 4. Group D animals received injections of physiological salineinstead of 3H-thymidine over the same time course as group A. Animalsfrom all groups were sacrificed by cervical dislocation 16-20 hoursfollowing the last series of injections. Brains were removed and neuraltissue obtained from the subependyma surrounding the lateral ventriclesin the forebrain was dissociated and the neural cells cultured asdescribed in Example 5. At 6 and 8 days in vitro, the total number ofspheres was counted in each of the 35 mm wells.

Control animals that received a series of saline injections formed thesame number of spheres as animals that received 3H-thymidine on day 0(which kills the normally proliferating subependymal cells). Thisindicates that the constitutively proliferating subependymal cells arenot the source of stem cells isolated in vitro. Animals that received asecond series of injections on day 2 formed 45% the number of spheres(similar to the number of proliferating subependymal cells observed invivo). When a second series of injections was done on day 4, the numberof spheres formed in vitro was not significantly different from controlvalues, again correlating with the in vivo findings. Taken together,this data indicates that the multipotent spheres, which are isolated invitro in the presence of EGF, are formed from the relatively quiescentstem cell population within the subependyma in vivo.

EXAMPLE 27 In Vivo Proliferation of Neural Stem Cells of LateralVentricle

A replication incompetent retrovirus containing the β-galactosidase gene[as described in Walsh and Cepko, Science 241:1342, (1988)] was injectedinto the forebrain lateral ventricles of CD1 adult male mice (25-30 gfrom Charles River). The injected retrovirus was harvested from the BAGcell line (ATCC CRL-9560) according to the method of Walsh and Cepko(supra). Mice were anesthetized using 65 mg/kg, i.p. sodiumpentobarbital. Unilateral stereotactic injections of 0.2-1.0 μl ofretrovirus were injected into the lateral ventricle using a 1 μlHamilton syringe. The coordinates for injection were AP +4.2 mm anteriorto lambda, L±0.7 mm, and DV -2.3 mm below dura, with the mouth bar at -2mm below the interaural line.

On the same day as, one day, or six days following the retrovirusinjection, an infusion cannulae attached to a 0.5 μl/hour ALZET osmoticmini-pumps filled with 3.3-330 μg/ml of EGF were surgically implantedinto the lateral ventricles at the identical stereotactic coordinates asstated above. The infusion cannula kits were obtained from ALZA. Theinfusion cannulae were cut to 2.7 mm below the pedestal. The pumps weresecured to the mouse skull by use of acrylic cement and a skull screwcontralateral and caudal to the injection site. The osmotic mini-pumpwas situated subcutaneously under and behind the armpit of the leftfront paw and connected to the infusion cannula by the means ofpolyethylene tubing.

Six days following initiation of EGF infusion the animals weresacrificed with an overdose of sodium pentobarbital. Mice weretranscardially perfused with 2% buffered paraformaldehyde, and thebrains were excised and post fixed overnight with 20% sucrose in 2%buffered paraformaldehyde. Coronal slices were prepared with -20 celsiuscryostat sectioning at 30 μm. Slices were developed for β-galhistochemistry as per Morshead and Van der Kooy (supra).

Under these conditions, regardless of the day post retrovirus injection,infusion of EGF resulted in an expansion of the population of β-gallabelled cells from an average of 20 cells per brain up to an average of150 cells per brain and the migration of these cells away from thelining of the lateral ventricles. Infusion of FGF-2 at 33 μg/ml resultedin an increase in the number of β-gal labelled cells, but this increasewas not accompanied by any additional migration. Infusion of EGF and FGFtogether resulted in an even greater expansion of the population ofβ-gal labelled cells from 20 cells per brain to an average of 350 cellsper brain.

These results indicate that FGF may be a survival factor for relativelyquiescent stem cells in the subependyma layer, whereas EGF may act as asurvival factor for the normally dying progeny of the constitutivelyproliferating population. The synergistic increase in β-galactosidasecell number when EGF and FGF are infused together further reflects thedirect association between the relatively quiescent stem cell and theconstitutively proliferating progenitor cell.

EXAMPLE 28 In Vivo Proliferation of Neural Stem Cells of the Third andFourth Ventricles and the Central Canal

A retroviral construct containing the β-galactosidase gene ismicroinjected (as in Example 27) into the III ventricle of thediencephalon, IV ventricle of the brain stem and central canal of thespinal cord. Minipumps containing EGF and FGF are then used tocontinuously administer growth factors for six days (as in Example 27)into the same portion of the ventricular system that the retroviralconstruct was administered. This produces an increase in the number ofβ-galactosidase producing cells which survive and migrate out into thetissue near the III ventricle, IV ventricle and central canal of thespinal cord forming new neurons and glia.

EXAMPLE 29 In Vivo Modification and Proliferation of Neural Stem Cellsand Differentiation of Neural Stem Cell Progeny of the Lateral Ventricle

A retroviral construct containing the TH gene as well as theβ-galactosidase gene is microinjected into the adult lateral ventricleas in Example 27. Minipumps containing EGF, FGF, or EGF and FGF togetherare then used to continuously administer the growth factor(s) into thelateral ventricle for 6 days as in Example 27. As the infectedsubependymal cells migrate out into the striatum they differentiate intoneuronal cells that produce dopamine as measured directly byimmunofluorescence with an antibody and (from a direct functional assay)by the ability to overcome the rotational bias produced by unilateral6-hydroxydopamine lesions.

EXAMPLE 30 In Vivo Infusion of Growth Factors into Ventricles to ObtainElevated Numbers of Neural Stem Cells

Adult male CD₁ albino mice (30-35g) from Charles River wereanaesthetized with sodium pentobarbital (0.40 mL of a 10% solution) andplaced in a stereotaxic apparatus. The dorsal aspect of the skull wasexposed with a longitudinal incision. Cannulas were inserted into thefourth ventricle (stereotaxic coordinates A/P -7.0, L±0.3 D/V -5.8),cerebral aqueduct (A/P -4.8 L±D/V -2.6), or central canal (D/V -1.5).The cannulae were attached with sterile tubing to subcutaneouspositioned ALZET osmotic mini-pumps containing 25 μg/mL EGF (Becton40001) and/or 25 μg/mL FGP-2 (R&D Systems 233-FB). Pumps containingsterile saline plus 0.1% mouse albumin (Sigma A3134) were used ascontrols. The incisions were closed with dental cement.

Six days following surgery mice were injected with 0.15 mL BrdU (SigmaB5002); 18 mg/mL in 0.007% NaOH/0.1M PBS) every 2 hours for 8 hours.They were killed 0.5 hours after the last injection with an anaestheticoverdose, and transcardially perfused with 10 mL of ice-cold sterilesaline followed by 10mL of ice-cold Bouin's fixative (5% glacial aceticacid, 9% formaldehyde, 70% picric acid). The cervical spinal cord regionwas dissected out and post-fixed overnight at 4° C. in Bouin'spost-fixative solution (9% formaldehyde, 70% picric acid). The followingday the tissue was cryoprotected by immersion in 10% sucrose for 2hours, 20% sucrose for 2 hours, and 30% sucrose overnight. The tissuewas frozen in powdered dry ice, mounted in Tissue-Tek (Miles 4583) at-18° C., and 30 μm serial sagittal sections were mounted onto gel-subbedglass slides. Each slide also contained one or more 30 μm coronalsections through the lateral ventricles from the brain of the sameanimal to serve as a positive control. Slides were kept at -80° C. untilprocessed. Immunohistochemistry: Slides were rinsed in PBS 3×15 minutesin 0.1M PBS at room temperature, hydrolyzed with 1N HCl for 60 minutesat 37° C., rinsed for 3×15 minutes in 0.1M PBS at room temperature,placed in 6% H₂ O₂ in methanol for 30 minutes at room temperature,rinsed for 3×15 minutes in 0.1M PBS at room temperature, and incubatedin 10% normal horse serum (Sigma H-0146) in 0. 1M PBS or 20 minutes atroom temperature. Slides were incubated overnight at room temperature inanti-BrdU monoclonal antibody (Becton 7580) that was diluted 1:50 in0.1M PBS containing 1.5% normal horse serum and 0.3% TRITON®. Thefollowing day the slides were rinsed in PBS for 3×10 minutes in 0.1M PBSat room temperature, incubated with biotinylated horse anti-mouse IgG(Vector BA-2000) for 2 hours at room temperature, rinsed for 3×15minutes in 0.1M PBS at room temperature, incubated in ABC reagent(Vector PK-6100) for 2 hours at room temperature, rinsed for 3×15minutes in 0.1M PBS at room temperature, and developed with DAB reagentfor 2 to 4 minutes. The slides were coverslipped with Aqua Polymount(Polysciences 18606). The number of BrdU positive cells was counted percervical spinal cord section. Some BrdU labelled cells were found in thesaline control sections. Treatment with either EGF or FGF-2 resulted ina significant increase in the number of BrdU labelled cells seencompared to control. The combination of EGF plus FGF-2 produced even agreater amount of BrdU positive cells per section.

EXAMPLE 31 In Vivo Infusion of Growth Factors into Ventricles toIncrease Yield of Neural Stem Cells That Proliferate In Vitro

EGF pumps were implanted as described in Example 27. Animals weresacrificed by cervical dislocation 6 days after the pump was implanted.Brains were removed and the stem cells isolated and counted as describedin Example 5.

Animals infused with EGF into the lateral ventricles for 6 days prior tosacrifice and brain culturing had 4 times as many spheres forming after9 days in vitro compared to control animals which received saline pumpsfor the same 6 day period. Thus, infusing EGF into the lateralventricles in vivo prior to removal and dissociation of neural tissue,greatly increases the yield of stem cells which proliferate and formneurospheres in vitro.

EGF and FGF can be infused into the ventricles to further increase theyield of neural stem cells obtainable from the neural tissue.Neurospheres generated by this method are used as a source of donorcells for later transplantation into degenerated areas of human adultCNS. Neurospheres can also be proliferated accordingly from a patient'sown CNS stem cells and transplanted back into the patient.

EXAMPLE 32 In Vivo Modification of Neural Cells with bcl-2 Gene

A retroviral construct containing the human bcl-2 gene and theα-galactosidase gene is microinjected into the adult mouse lateralventricle. A control mouse is injected with a retroviral constructcontaining only the β-galactosidase gene. One of the two progeny of eachof the constitutively proliferating subependymal cells of the adultlateral ventricle normally dies within a few hours after division. Thebcl-2 gene product prevents the programmed death of cells in severalother tissues. In the adult subependyma, single cells infected with boththe β-galactosidase and bcl-2 genes are marked by expression of boththese gene products. These cells are identified in brain tissue sliceswith antibodies specific to β-galactosidase and human Bcl-2.Proliferating infected subependymal cells so infected produce largernumbers of cells per clone relative to the control. Thus, Bcl-2 inducesthe survival of the one normally dying progeny of each division of aconstitutively proliferating adult subependymal cell. Moreover, thebcl-2 infected progeny migrate out into striatal and septal tissue toproduce new neurons and glia. This indicates that EGF and Bcl-2 act as asurvival factors for the normally dying progeny of constitutivelyproliferating adult subependymal cells which generate new neurons andglia in vivo.

EXAMPLE 33 In Vivo Modification of Neural Cells with NGF Gene

A retroviral construct containing the NGF gene is microinjected usingthe procedure described in Example 27 to infect the constitutivelyproliferating adult subependymal cells of the lateral ventricle. Thus,these cells are used to produce an endogenous growth factor in the adultbrain. Levels of NGF produced by the transfected cells are measureddirectly by radioimmunoassay and (from a direct functional assay) byrescue of basal forebrain cholinergic neurons in vivo after axotomyinjury in the model developed by Gage and collaborators (P.N.A.S.83:9231, 1986).

EXAMPLE 34 Generation of Dopamine Cells in the Striatum by theAdministration of a Composition Comprising Growth Factors to the LateralVentricle

Adult male CD₁ mice were anesthetized and placed in a stereotaxicapparatus. A cannula, attached to an ALZET minipump, was implanted intoa lateral ventricle of each animal. The minipumps were subcutaneouslyimplanted and were used to deliver (a) conditioned medium (from the ratB49 glial cell line, obtained from D Schubert, Salk Institute) plus bFGF(R&D Systems, 25 μg/ml) plus heparan sulfate (Sigma, 10 IU/ml) (CMF) or(b) EGF (Chiron, 25 μg/ml) plus bFGF (25 μg/ml) plus heparan sulfate (10IU/ml) plus 25% FBS (E+F+FBS) or (c) sterile saline solution (SAL) as acontrol, into the lateral ventricles. Once batch of animals wassacrificed one day after completion of the delivery regimen and theothers were sacrificed twenty days later. The subventricular zones(SVZs) of these mice were dissected out, separating the cannulated, andtherefore treated, side from the noncannulated control sides. Thesubstantia nigra (SN) region of these mice were also recovered. TotalRNA was extracted from these tissues using the guanidium thiocyanateacid phenol method [Chomzynski and Sacchi, Annal. Biochem. 162: 156-159,(1987)]. The RNA was then reverse transcribed to produce cDNA. ThesecDNAs were subject to PCR using primers designed to bracket a 254nucleotide region of the TH messenger RNA (mRNA) and thermal cyclingconditions favoring quantitative amplification. The PCR products wereelectrophoresed on a 2% agarose gel and then capillary blotted onto apositively charged nylon membrane. Radioactively labelled cDNA probe toTH was hybridized to the filter and detected by autoradiography. Theautoradiograph was scanned and analyzed by densitometry to obtainrelative levels of mRNA for TH in the SVZs of the cannulated sides inresponse to the treatments in the non-cannulated control SVZs and in theSN. In animals analyzed one day after treatment, the administration ofE+F+FBS produced an eleven-fold increase in the level of TH mRNA in theSVZ compared to that observed in response to CMF, which in turn was morethan twice the level seen with SAL. Twenty one days after treatment, theamount of TH mRNA detected in response to treatment with E+F+FBS wasapproximately the same as that detected after one day, while CMF and SALtreated SVZs had TH mRNA levels which were below detectable limits andwere indistinguishable from the non-cannulated SVZ controls. Under alltreatments, the SN had measurable amounts of TH mRNA.

EXAMPLE 35 Detection of Dopaminergic Cells in Striatal Tissue Using DualLabeling

Male CD₁ mice (Charles River, approximately 4 to 6 weeks old) were givenintraperitoneal injections of BrdU (Sigma, 120 mg/kg) at 2 hourintervals over a 24 hour period, in order to label mitotically activecells. A cannula attached to an ALZET minipump was then implantedunilaterally into a lateral ventricle of each animal in order to delivercompositions a-c (CMF, E+F+FBS, or sterile saline) described in Example34.

Animals were sacrificed 24 hours after the administration of growthfactors using a lethal dose of pentobarbital anesthetic. The animalswere then perfused through the heart with 10 ml of ice could 4%paraformaldehyde solution. The brains were removed and tissue in theregion extending from the olfactory bulb to the third ventricle,including the striatum, was dissected out and stored overnight at 4° C.in a 30% sucrose/4% paraformaldehyde solution. The tissue was thenfrozen on dry ice and kept at -70° C. until processed. 30 μm coronalsections were cut using a cryostat and the sections were placed in 12well porcelain dishes, to which 400 μl of PBS had been added. Sectionswere rinsed with fresh PBS and incubated overnight with the followingprimary antibodies: anti-TH (rabbit polyclonal, 1:1000, Eugene TechInternational Inc.; or 1:100, Pel-freeze) and mouse anti-BrdU (1:55,Amersham), prepared in PBS/10% normal goat serum/0.3 TRITON® X-100.Following three rinses in PBS, goat anti-rabbit rhodamine and goatanti-mouse fluorescein (Jackson) were applied in PBS for 50 minutes atroom temperature. Sections were then washed three times (10 minuteseach) in PBS, placed on glass slides, dried and then coverslipped usingFluorsave (Calbiochem #345789).

The location of dopaminergic neurons was determined by mapping thelocation of TH-immunoreactive (TH+) cells, or TH+ and BrdU+ cells inrelation to the ventricles. In response to saline injections made intothe lateral ventricles, the normal population of TH+ fibers weredetected in the striatum but no TH+ cell bodies were detected in thisregion. CMF treatment resulted in the detection of TH+ cell bodies, inaddition to the normal population of TH+ fibers, in the striatum and inthe region of the third ventricle. E+F+FBS treatment had the mostprofound effect resulting in the detection of the most TH+ cell bodies.Several of the TH+ cell bodies were also BrdU positive.

EXAMPLE 36 Rat Model of Parkinson's Disease Measures the Effects of InVivo Administration of Growth Factors

The 6-OHDA lesion rat model of Parkinson's disease is used to measurethe effects of administering various combinations of growth factors tothe lateral ventricle. Unilateral 6-OHDA lesions are performed in therat model and rotation behavior is observed. Minipumps aresubcutaneously implanted into the animals as described in Example 34.EGF (Chiron, 25 μg/ml) plus bFGF (25 μg/ml) plus heparan sulfate (10IU/ml) plus 25% FBS is continuously administered to the lateralventricle. Saline is administered to control animals. The ability toovercome the rotational bias produced by the unilateral 6-OHDA lesionsis observed.

EXAMPLE 37 Screening of Drugs or Other Biological Agents for Effects onMultipotent Neural Stem Cells and Neural Stem Cell Progeny

A. Effects of BDNF on Neuronal and Glial Cell Differentiation andSurvival

Precursor cells were propagated as described in Example 4 anddifferentiated using Paradigm 3 described in Example 7. At the time ofplating the EGF-generated cells, BDNF was added at a concentration of 10ng/ml. At 3, 7, 14, and 21 days in vitro (DIV), cells were processed forindirect immunocytochemistry. BrdU labeling was used to monitorproliferation of the precursor cells. The effects of BDNF on neurons,oligodendrocytes and astrocytes were assayed by probing the cultureswith antibodies that recognize antigens found on neurons (MAP-2, NSE,NF), oligodendrocytes (O4, GalC, MBP) or astrocytes (GFAP). Cellsurvival was determined by counting the number of immunoreactive cellsat each time point and morphological observations were made. BDNFsignificantly increased the differentiation and survival of neurons overthe number observed under control conditions. Astrocyte andoligodendrocyte numbers were not significantly altered from controlvalues.

B. Effects of BDNF on the Differentiation of Neural Phenotypes

Cells treated with BDNF according to the methods described in Part Awere probed with antibodies that recognize neural transmitters orenzymes involved in the synthesis of neural transmitters. These includedTH, ChAT, substance P, GABA, somatostatin, and glutamate. In bothcontrol and BDNF-treated culture conditions, neurons tested positive forthe presence of substance P and GABA. As well as an increase in numbers,neurons grown in BDNF showed a dramatic increase in neurite extensionand branching when compared with control examples.

C. Identification of Growth-Factor Responsive Cells

Cells that are responsive to growth factor treatment were identified bydifferentiating the EGF-generated progeny as described in Example 7,paradigm 3 and at 1 DIV adding approximately 100 ng/ml of BDNF. At 1, 3,6, 12 and 24 hours after the addition of BDNF the cells were fixed andprocessed for dual label immunocytochemistry. Antibodies that recognizeneurons (MAP-2, NSE, NF), oligodendrocytes (O4, GalC, MBP) or astrocytes(GFAP) were used in combination with an antibody that recognizes c-fosand/or other immediate early genes. Exposure to BDNF results in aselective increase in the expression of c-fos in neuronal cells.

D. Effects of BDNF on the Expression of Markers and Regulatory FactorsDuring Proliferation and Differentiation

Cells treated with BDNF according to the methods described in Part A areprocessed for analysis of the expression of FGF-R1, as described inExample 39 or other markers and regulatory factors, as described inExample 40.

E. Effects of BDNF administration During Differentiation on theElectrophysiological Properties of Neurons

Neurons treated with BDNF during differentiation, according to themethods described in Part A, are processed for the determination oftheir electrophysiological properties, as described in Example 41.

F. Effects of Chlorpromazine on the Proliferation, Differentiation, andSurvival of Growth Factor Generated Stem Cell Progeny

Chlorpromazine, a drug widely used in the treatment of psychiatricillness, is used in concentrations ranging from 10 ng/ml to 1000 ng/mlin place of BDNF in Examples 7A to 7E above. The effects of the drug atvarious concentrations on stem cell proliferation and on stem cellprogeny differentiation and survival is monitored. Alterations in geneexpression and electrophysiological properties of differentiated neuronsare determined.

EXAMPLE 38 Stem Cell Proliferation Assay

Primary cells were obtained from E14 mice and prepared as detailed inExamples 1 and 4. Either EGF, EGF and FGF or EGF and BMP-2 were added tocomplete medium at a concentration of 20 ng/ml of each growth factor,with the exception of BMP-2 which was added at a concentration of 10ng/ml. Cells were diluted with one of the prepared growthfactor-containing media to a concentration of 25,000 cells/ml. 200 μl ofthe cell/medium combination were pipetted into each well of a 96-wellplace (Nuclon) with no substrate pretreatment. Cells were incubatedunder the same conditions as outlined in Example 4.

After 8-10 DIV the number of neurospheres was counted and the resultstabulated. As cells grown in a combination of EGF and FGF producedsignificantly more neurospheres than cells grown in the presence of EGFalone. The combination of EGF and BMP-2 inhibited neurospheredevelopment.

EXAMPLE 39 Comparison of Receptor and Growth Factor Expression inUndifferentiated vs. Differentiated Stem Cell-Derived Progeny by ReverseTranscription-Polymerase Chain Reaction (RT-PCR)

Neurospheres were generated as described in Example 4, and some weredifferentiated as per Paradigm 1, Example 7. RNA from eitherundifferentiated or differentiated neurospheres was isolated accordingto the guanidinium thiocyanate acid phenol procedure of Chomzynski andSacchi -Anal. Biochem. 162: 156-159 1987)]. Complementary DNA (cDNA) wassynthesized from total RNA using reverse transcriptase primed with oligodT. Gene-specific primers were designed and synthesized and theseprimers were used in PCR to amplify cDNAs for different growth factorsand growth factor receptors. Amplified material was run on agarose gelsalongside molecular weight markers to ensure that PCR products were ofthe expected size, while the identity of PCR fragments was confirmed byrestriction enzyme analysis and by sequencing [Arcellana-Panlilio,Methods Enzymol. 225: 303-328 (1993)]. An ethidium-stained agarose gelvisualized via UV transillumination showed the detection of three growthfactor receptor transcripts, namely EGF-R, FGF-R, and LIF-R, inundifferentiated and differentiated stem cell-derived progeny. Table Ilists the primer sets analyzed and the results of undifferentiated anddifferentiated cells.

                  TABLE I                                                         ______________________________________                                        Primer Sets Analyzed                                                                           Undifferentiated                                                                          Differentiated                                     Cells Cells                                                                 ______________________________________                                        Actin            +           +                                                  NGF + nd                                                                      EGFr.sup.m + +                                                                bFGFr + +                                                                     LIFr.sup.m + +                                                                tyrosine hydroxylase + +                                                      choline acetyltransferase.sup.m nd +                                          cholecystokinin.sup.m nd -                                                    enkephalin.sup.m nd +                                                         tyrosine kinase-rA + +                                                        tyrosine kinase-rB + +++++                                                    tyrosine kinase-rC + +                                                      ______________________________________                                         r = receptor                                                                  m = derived from mouse                                                        nd = no data available                                                   

EXAMPLE 40 Isolation of Novel Markers and Regulatory Factors Involved inNeural Stem Cell Proliferation and Differentiation

Neurospheres are generated as described in Example 4 using CNS tissuefrom CD₁ albino mice (Charles River). Some of these neurospheres areallowed to differentiate according to the rapid differentiation paradigmof Example 7 producing cultures enriched in neurons, astrocytes, andoligodendrocytes. Total RNA is extracted from the undifferentiatedneurospheres as well as the differentiated cell cultures using theguanidinium thiocyanate acid phenol method referred to in Example 39.Messenger RNA (mRNA) is isolated by exploiting the affinity of its polyA tract to stretches of either U's or T's. Reverse transcription of themRNA produced cDNA, is then used to make primary libraries in eitherplasmid [Rothstein et al., Methods in Enzymology 225:587-610 (1993)] orlambda phage vectors. To isolate cDNAs that are specific to eitherundifferentiated or differentiated stem cell derived progeny, cDNA fromone is hybridized to RNA from the other, and vice versa. Theunhybridized, and thus culture type-specific, cDNAs in each case arethen used to construct subtracted libraries [Lopez-Fernandez and delMazo, Biotechniques 15(4):654-658 (1993)], or used to screen the primarylibraries.

Stem cell-derived undifferentiated cell specific and differentiated cellspecific cDNA libraries provide a source of clones for novel markers andregulatory factors involved in CNS stem cell proliferation anddifferentiation. Specific cDNAs are studied by sequencing analysis todetect specific sequence motifs as clues to identity or function, anddatabase searching for homologies to known transcripts. Using cDNAs in ahybridization to various RNA samples electrophoresed on anagarose-formaldehyde gel and transferred to a nylon membrane, allows theestimation of size, relative abundance, and specificity of transcripts.All or portions of cDNA sequences are used to screen other libraries inorder to obtain either complete mRNA sequences or genomic sequenceinformation. Antibodies directed against fusion proteins generated fromspecific cDNAs are used to detect proteins specific to a particular cellpopulation, either by immunocytochemistry or by Western Blot analysis.Specific gene sequences are used to isolate proteins that interact withputative regulatory elements that control gene expression. Theseregulatory elements are then used to drive the expression of anexogenous gene, such as beta-galactosidase.

EXAMPLE 41 Electrophysiological Analysis of Neurons Generated FromGrowth Factor-Responsive Stem Cells and Exposed to a Biological Agent

Neurospheres were generated as described in Example 4. Neurospheres weredissociated using the technique described in paradigm 2, Example 7. Theclonally derived cells were plated at low density and differentiated inthe presence of bFGF. The electrophysiological properties of cells withthe morphological appearance of neurons were determined as described asdescribed by Vescovi et al. [Neuron, 11: 951-966 (1993)]. Under wholecell current clamp, the mean resting potential and input resistance were-62±9 mV and 372±MΩ. Rectangular suprathreshold current steps, (-100 pA)elicited regenerative potential responses in which the amplitude andtime course were stimulus dependent. After the completion ofelectrophysiological experiments, the cell morphology was visualized byintracellular excitation of 5-carboxyfluorescein.

EXAMPLE 42 Screening for the Effects of Drugs or Other Biological Agentson Growth Factor-Responsive Stem Cell Progeny Generated From TissueObtained From a Patient with a Neurological Disorder

The effects of BDNF on the EGF-responsive stem cell progeny generatedfrom CNS tissue obtained at biopsy from a patient with Huntington'sdisease is determined using the methods outlined in Example 7, A to E.BDNF is a potent differentiation factor for GABAergic neurons andpromotes extensive neuronal outgrowth. Huntington's Disease ischaracterized by the loss of GABAergic neurons (amongst others) from thestriatum.

EXAMPLE 43 Assay of striatum-derived neurosphere proliferation inresponse to various combinations of proliferative and regulatory factors

Paradigm 1:

Primary striatal cells prepared as outlined in Example 1 were suspendedin Complete Medium, without growth factors, plated in 96 well plates(Nunclon) and incubated as described in Example 4. Following a one hourincubation period, a specific proliferative factor, or a combination ofproliferative factors including EGF, or bFGF (recombinant human bFGF: R& D Systems), or a combination of EGF and bFGF, or EGF plus FGF plusheparan sulfate (Sigma), or bFGF plus heparan sulfate made up inComplete Medium at a concentration of 20 ng/ml for each of the growthfactors and 2 μg/ml for heparan sulfate), was added to each well of theplate.

Activin, BMP-2, TGF-β, IL-2, IL-6, IL-8, MIP-1∂, MIP-1β, MIP-2 (allobtained from Chiron Corp.), TNFα, NGF (Sigma), PDGF (R&D Systems), EGFand CNTF (R. Dunn and P. Richardson, McGill University) were made up inseparate flasks of compete medium to a final concentration of 0.2 μg/ml.Retinoic acid (Sigma) was added at a concentration of 10-6 M. 10 μl ofone of these regulatory factor-containing solutions was added to eachproliferative factor-containing well of the 96 well plates. Controlwells, containing only proliferative factors, were also prepared.

In another set of experiments, the neurosphere inducing properties ofeach of these regulatory factors was tested by growing cells in theirpresence, in proliferative factor-free Complete Medium. None of theseregulatory factors, with the exception of EGF, when used in the absenceof a proliferation-inducing factor such as EGF or FGF, has an effect onneural stem cell proliferation.

The activin, BMP-2, TGF-β. IL-2, IL-6, IL-8, MIP-1∂, MIP-1β, MIP-2, TNFαand EGF additions were repeated every second day, CNTF which was addedeach day and retinoic acid, NGF and PDGF were added only once, at thebeginning of the experiment. The cells were incubated for a period of10-12 days. The number of neurospheres in each well was counted and theresulting counts tabulated using Cricket Graph III. Other relevantinformation regarding sphere size and shape were also noted.

In general, bFGF had a greater proliferative effect than EGF on thenumbers of neurospheres generated per well. In the presence of 20 ng/mlEGF, approximately 29 neurospheres per well were generated. In thepresence of bFGF, approximately 70 neurospheres were generated. However,in bFGF alone, the neurospheres were only about 20% of the size of thosegenerated in the presence of EGF. The combination of EGF and bFGFproduces significantly more neurospheres than does EGF alone, but fewerthan seen with bFGF alone. The neurospheres are larger than those seenin bFGF alone, approximating those seen in EGF. In the case of bFGFgenerated spheres, the addition of heparan sulfate increased the size ofthe spheres to about 70% of the size of those which occur in response toEGF. These data suggest that EGF and FGF have different actions withrespect to the induction of stem cell mitogenesis.

The effects of the regulatory factors added to the proliferativefactor-containing wells are summarized in Table II. In general, the TGFBfamily, interleukins, macrophage-inhibitory proteins, PDGF, TNFα,retinoic acid (10⁻⁶ M) and CNTF significantly reduced the numbers ofneurospheres generated in all of the proliferative factors orcombinations of proliferative factors tested. BMP-2 (at a dose of 10ng/ml), completely abolished neurosphere proliferation in response toEGF. EGF and heparan sulfate both greatly increased the size of theneurospheres formed in response to bFGF (about 400%).

                                      TABLE II                                    __________________________________________________________________________    PROLIFERATIVE FACTORS                                                                      EGF    bFGF  EGF + bFGF                                                                          bFGF + Heparan                                                                        EGF + bFGF + Heparan                  REGULATORY FACTORS                                                                         #   size                                                                             #  size                                                                             #  size                                                                             #   size                                                                              #     size                            __________________________________________________________________________    TGFβ Family ♦                                                           -57%                                                                              -- -57%                                                                             -- -34%                                                                                -55%                                                                              --  -20%  --                                BMP-2 -100% n/a -5% = +16%                                                    -3% -- +10%                                                                   Interleukins -21% = -23% = -37% -- -28% = -39% --                             MIP Family -25% = -6% = -32% -- -22% = -33% --                                NGF -10% = 0% = -30% = +5% = -48% =                                           PDGF -1.5% = -4% = -26% = -10% = -27% =                                       TNFα -17% = -17% = -41% = -21% = -37% =                                 10.sup.-6 M Retinoic Acid -8%                                                 -61% -- -31%                                                                  -65%                                                                          -45%                                                                          CNTF -23% -- -77% ˜ -81%                                                -81% -- -84%                                                                  EGF --  -14% ++ --  -17% = --                                                 Heparan Sulfate 0% = 0% ++ 0% =                                             __________________________________________________________________________     ♦Excluding BMP2 (i.e. TGFα and activin)                   Numbers of neurospheres generated (#) are given as percentages that           reflect the decrease (-) or increase (+) in numbers of neurospheres per       well, in response to a PROLIFERATIVE FACTOR in the presence of a              REGULATORY FACTOR, compared with the number of neurospheres proliferated      in the absence of the REGULATORY FACTOR.                                      Size of neurospheres generated in the presence of PROLIFERATIVE FACTORS       and REGULATORY FACTORS compared to those generated in the presence of         PROLIFERATIVE FACTORS alone are indicated as follows:                         ++: much larger; +: larger; =: approximately the same size; ˜:          variable in size; --: smaller; -: much smaller                                Paradigm 2: Antisense/sense experiments: Embryonic tissue was prepared as     outlined in Example 1 and plated into 96 well plates in Complete Medium. 

Antisense and sense experiments were carried out using the followingoligodeoxynucleotides (all sequences written 5' to 3'):

    EGF receptor:                                                                          Sense strand:                                                                            GAGATGCGACCCTCAGGGAC                                                                       (SEQ ID NO:1)                                   -  Antisense strand:GTCCCTGAGGGTCGCATCTC (SEQ ID NO:2)                        - EGF: Sense strand:TAAATAAAAGATGCCCTGG (SEQ ID NO:3)                         -  Antisense strand: CCAGGGCATCTTTTATTTA (SEQ ID NO:4)                 

Each oligodeoxynucleotide was brought up and diluted in ddH₂ O and keptat -20° C. Each well of the 96 well plates received 10 μl ofoligodeoxynucleotide to give a final concentration of either 1, 2, 3, 4,5, 10 or 25 μM. Oligodeoxynucleotides were added every 24 hours. The EGFreceptor (EGFr) and EGF oligodeoxynucleotides were applied to culturesgrown in bFGF (20 ng/ml), and EGFr oligodeoxynucleotides were applied tocultures grown in EGF (20 ng/ml). Cells were incubated at 37° C., in a5% CO₂ 100% humidity incubator. After a period of 10 to 12 days, thenumber of neurospheres per well was counted and tabulated. Aconcentration of 3 μM of antisense oligodeoxynucleotides produced a 50%reduction in the number of neurospheres generated per well, whereas thesense oligodeoxynucleotides had no effect on the number of neurospheresgenerated in response to EGF and FGF. Both the sense and antisenseoligodeoxynucleotides were toxic to cells when 10 μM or higherconcentrations were used.

Similar experiments can be performed using the followingoligonucleotides:

    FGF receptor:                                                                          Sense strand:                                                                            GAACTGGGATGTGGGGCTGG                                                                       (SEQ ID NO:5)                                   -  Antisense strand:CCAGCCCCACATCCCAGTTC (SEQ ID NO:6)                        - FGF: Sense strand: GCCAGCGGCATCACCTCG (SEQ ID NO:7)                         - Antisense strand:CGAGGTGATGCCGCTGGC (SEQ ID NO:8)                    

The FGF receptor (FGFr) and FGF oligodeoxynucleotides are applied tocultures grown in EGF, and PGFr oligodeoxynucleotides are applied tocultures grown in bFGF.

Paradigm 3:

Embryonic tissue is prepared as outlined in Example 1 and plated into 96well plates. Complete Medium, containing 20 ng/ml of either EGF of bFGFis added to each well. 10 μl of diluted phorbol 12-myristate 13 acetate(PMA) is added once, at the beginning of the experiment, to each well ofthe 96 well plates, using an Eppendorf repeat pipetter with a 500 μl tipto give a final concentration of either 10, 20, 40, 100 or 200 μg/ml.Cells are incubated at 37° C. in a 5% CO₂ 100% humidity incubator. Aftera period of 10 to 12 days the number of neurospheres per well is countedand tabulated.

Paradigm 4:

Embryonic tissue is prepared as outlined in Example 1 and plated into 96well plates. 10 μl of diluted staurosporine is added to each well of a96 well plate, using an Eppendorf repeat pipetter with a 500 μl tip togive a final concentration of either 10, 1, 0.1, or 0.001 μM ofstaurosporine. Cells are incubated at 37° C., in a 5% CO₂ 100% humidityincubator. After a period of 10 to 12 days, the number of neurospheresper well is counted and tabulated.

EXAMPLE 44: Adult Spinal Cord Stem Cell Proliferation--In VitroResponses to Specific Biological Factors or Combinations of Factors

Spinal cord tissue was removed from 6 week to 6 month old mice, asfollows: cervical tissue was removed from the vertebral column regionrostral to the first rib; thoracic spinal tissue was obtained from theregion caudal to the first rib and approximately 5 mm rostral to thelast rib; lumbar-sacral tissue constituted the remainder of the spinalcord. The dissected tissue was washed in regular artificialcerebrospinal fluid (aCSF), chopped into small pieces and then placedinto a spinner flask containing oxygenated aCSF with high Mg²⁺ and lowCa²⁺ and a trypsin/hyaluronidase and kynurenic acid enzyme mix tofacilitate dissociation of the tissue. The tissue was oxygenated,stirred and heated at 30° C. for 1 1/2 hours, then transferred to a vialfor treatment with a trypsin inhibitor in media solution(DMEM/12/hormone mix). The tissue was triturated 25-50 times with a firenarrow polished pipette. The dissociated cells were centrifuged at 400r.p.m. for 5 minutes and then resuspended in fresh media solution. Cellswere plated in 35 mm dishes (Costar) and allowed to settle. Most of themedia was aspirated and fresh media was added. EGF alone, or EGF andbFGF were added to some of the dishes to give a final concentration of20 ng/ml each, and bFGF (20 ng/ml) was added, together with 2 μg/ml ofheparan sulfate, to the remainder of the dishes. The cells wereincubated in 5% CO₂, 100% humidity, at 37° C. for 10-14 days. Thenumbers of neurospheres generated per well were counted and the resultstabulated. EGF alone resulted in the generation of no neurospheres fromany of the spinal cord regions. In the presence of EGF plus bFGF,neurospheres were generated from all regions of the spinal cord, inparticular the lumbar sacral region. The combinations of EGF+FGF andFGF+ heparan sulfate produced similar numbers of spheres in the cervicalregion, whereas the combination of bFGF plus heparan sulfate resulted infewer neurospheres from the thoracic and lumbar regions.

EXAMPLE 45 Transplantation of Multipotent Neural Stem Cell Progeny inAnimal Models

I. TRANSPLANTATION PROCEDURE

1. Neurosphere preparation

Neural tissue was obtained from normal embryonic or adult CD1 mice andfrom embryonic or adult Rosa 26 mice (transgenic animals derived fromC57/BL/6 mice, which express the β-galactosidase gene in all cells, thusallowing the transplanted cells to be easily detected in host tissue).Neurospheres were generated using the procedures described in Examples1-5, passaged 2 to 8 times (see Example 6), and maintained in culturefor 6-10 days after the last passage.

2. Labeling and Preparation of Neural Stem Cell Progeny

16 hours prior to transplantation, neurospheres derived from embryonicand adult tissue were labeled with BrdU by adding BrdU to the media fora total concentration of 1 μM and/or with fluorescent latex beads(Polysciences; 1:100 dilution of 0.75 μm beads). Neurospheres weredetached from the substrate by gentle shaking, poured into 50 mlcentrifuge tubes and spun down (5 minutes, 400 r.p.m., 15° C., no brake)to remove the proliferation-inducing media used for the proliferationculture. The neurospheres derived from embryonic tissue were then washedtwice in Hank's buffered salt solution (HBSS), resuspended in 2 ml HBSSand dissociated by trituration (spheres drawn into a fire-polishedpasteur pipette 40×). The neurospheres derived from adult tissue weretrypsinized (0.05% in EDTA media; 5-10 min) and then a trypsin inhibitor(ovomucoid; 0.7-1.0 mg/ml in media) was added. The tubes were swirledand the neurospheres were recentrifuged (400 r.p.m., 15° C., no brake).Cells were resuspended in 2ml media (DMEM F12/hormone mix) anddissociated by mechanical trituration (25×). Live and dead cellsobtained from neurospheres derived from embryonic and adult tissue werecounted prior to being centrifuged to remove dead cells (10 min., 400r.p.m., 15° C., no brake). The live cells were resuspended toappropriate cell density (1-50×10⁶ cells/ml). The cells were recountedto determine the number of live and dead cells and cell viability wascalculated. The cells were then transferred to a microcentrifuge tubefor storage on ice prior to transplantation. When ready for use, cellswere resuspended prior to each cell injection by drawing cells into aneppendorf pipette tip (200 or 1000 μl).

3. Transplantation of Neural Stem Cell Progeny

The donor neural stem cell progeny were transplanted into selected sitesin the brain of normal, healthy neonate or adult CD1 or C57BL/6 mice oradult Wistar or Sprague-Dawley rats. In some cases, embryonic cells fromCD1 mice received in vitro gene transfer procedures prior totransplantation of the cells. The host animals were anaesthetized withsodium pentobarbital (65 mg/Kg) and placed into a stereotaxic apparatus.A skin incision was made to expose the surface of the skull orvertebrae. Injection sites were located using stereotaxic coordinates tolocate the desired site. Burr holes were drilled in the skull andvertebrae at the coordinate sites. A 5 μl syringe was housed on asyringe pump and attached to a stainless steel cannula (30-31 gauge) viaa short length of polyethylene tubing. A small air bubble and then 4-5μl of the desired cell suspension was drawn into the cannula. Thecannula was lowered to the desired location and 1-3 μl of the cellsuspension was injected at a speed of 0.1-0.5 μl/min. Animals thatreceived xenografts or allograft were treated with 0.1 mg/ml cyclosporinA in the drinking water to reduce the risk of tissue rejection.

4. Analysis of Transplanted Neural Stem Cell Progeny

The animals were allowed to survive for 2-12 weeks prior to sacrifice.At a specified time after transplantation, animals were perfusedtranscardially for aldehyde fixation of the brain and spinal cordtissue. A low-high pH perfusion protocol was used (Sloviter & Nilaver,(1987) Brain Res. Vol. 330:358-363). After perfusion, brains and spinalcords were removed, post-fixed, and then cryoprotected in sucrose/PBSfor cutting in a cryostat. Sections of tissue (10 μM) were cut andmounted on microscope slides directly in a sequential way so thatadjacent sections could be examined with different anatomical protocols.

Survival of transplanted cells labeled with fluorescent beads wereidentified by the localization of fluorescent beads within the cellcytoplasm. BrdU labeled cells (cells that had incorporated BrdU intotheir DNA during cell division in culture prior to transplantation) wereidentified using antibodies against BrdU (1:250-500;Monoclonal-Sera-lab; Polyclonal-Accurate Chem. & Sci). Antibodiesagainst GFAP (1:250 Monoclona-Boehringer, Polyclonal-BTI), or NeuN(1:250-500; Monoclonal-R. J. Mullen) were then used to identify thedifferentiation of the transplanted cells. Cell transplants derived fromtransgenic animals expressing β-galactosidase were histochemicallyanalyzed using methodology described by Turner and Cepko (1987) (Nature328:131-136)and by immunohistochemical staining. For Rosa 26 cells,antibodies against β-galactosidase were used to identify thetransplanted cells and antibodies to NeuN were used to identify cellsthat had differentiated into neurons. Human cells were identified withHLA antibodies (1:250, Monoclonal-Sera-labs). Antibodies were incubatedwith the tissue samples and detected using standard immunohistochemicalprotocols.

The results obtained from the animal models described below aresummarized in Tables III-V.

A. MODEL OF HUNTINGTON'S DISEASE

Rats were anesthetized with nembutal (25 mg/kg i.p) and injected withatropine sulfate (2 mg/kg i.p.). Animals sustained an ibotenate lesionof the striatum, stimulating Huntington's Disease in the animals. 7 daysafter the lesion, the animals received an injection of cells prepared asin Examples 1-5 under stereotaxic control. Injections were made to thelesioned area via a 21-gauge cannula fitted with a teflon catheter to amicroinjector. Injected cells were labelled with fluorescein-labelledmicrospheres. Animals were given behavioral tests before the lesion,after the lesion, and at various intervals after the transplant todetermine the functionality of the grafted cells at variouspostoperative time points, then killed and perfused transcardially with4% buffered paraformaldehyde, 0.1% glutaraldehyde and 5% sucrosesolution at 4 C. The brains were frozen in liquid nitrogen and stored at-20° C. until use. Brains sections were sliced to 26 μm on a cryostat,fixed in 4% paraformaldehyde and stained using the M6 monoclonalantibody to stain for mouse neurons, and then reacted with a secondaryanti-rat fluorescein-conjugated antibody. Neuronal and glial phenotypewas identified by dual labeling of the cells with antibody to NSE andGFAP.

B. PARKINSON'S DISEASE

Two animal models of Parkinson's Disease were used. In the first model,unilateral dopamine neurons of the substantia nigra were lesioned by thestereotaxic administration of 6-OHDA into the substantia nigra in adultCD 1 (1-4 μg) and C57BL/6 mice (1 μg), and Wistar rats (16 μg). Micewere pretreated with desipramine (25 mg/Kg i.p.) and rats werepretreated with pargyline (50 mg/Kg i.p.) both of which prevent theaction of 6-OHDA on noradrenergic neurons and allow the selectivedestruction of dopaminergic neurons. In one series of experiments,multipotent neural stem cell progeny obtained from embryonic Rosa 26mice, were prepared using the procedures described in Examples 1 and 4.The neural stem cell progeny were labeled, prepared, and transplantedinto the striatum of the lesioned C57BL/6 mice using the methodsdescribed above in this Example.

In a second series of experiments, the cells were administered to thesame regions in the brains of adult 6-OHDA Wister rats. In a thirdseries of experiments, proliferated fetal human cells (prepared asoutlined in Example 9), were transplanted into the striatum of the6-OHDA lesioned CD1 mice. After a survival period of 2 weeks, the hostanimals were sacrificed and their brains removed. The brain tissue wastreated and analyzed as described above.

The second animal model used was the adult mutant Weaver mice (JacksonLabs, 3-1/2 months), in which approximately 70% of the dopaminergicneurons of the substantia nigra are lost by the age of 3 months. Animalswere anaesthetized and the proliferated progeny of multipotent neuralstem cells derived from embryonic Rosa 26 mice were injected into thestriatal region of the animals according to the methods described above.The animals were allowed to survive for 15 days prior to sacrifice andanalysis of striatal tissue.

C. CARDIAC ARREST

Transient forebrain ischemia was induced in adult Wistar rats bycombining bilateral carotid occlusion with hypovolemic hypotension(Smith et al. (1984) Acta Neurol Scand 69:385-401). These procedureslesion the CA1 hippocampal pyramidal cells which is typical of damageobserved in humans following cardiac arrest and the cause of severememory and cognitive deficits. The progeny of proliferated multipotentneural stem cells, derived from embryonic Rosa 26 mice, were prepared asdescribed above and transplanted into the lesioned hippocampal of theischemia lesioned rats. After 8 days, the animals were sacrificed andtheir brains were removed and analyzed. β-gal positive cells, indicatingsurviving cells from the Rosa 26 donor) were detected in the lesionedhippocampal region. In addition, double labeled β-gal/NeuN+ cells werefound indicating that transplanted cells had differentiated intoneurons.

D. STROKE

Occlusion of the carotid arteries precipitates the occurrence ofischemic damage similar to that which occurs during stroke. Adult Wistarrats, in which the middle cerebral artery has been occluded to producesymptomatic lesions in the caudal striatum and parietal cortex, haveneural stem cell progeny implanted into the lesioned areas. After asurvival period, the animals are tested for behavioral improvements andare then sacrificed and their brains analyzed.

E. EPILEPSY

Implantation of an electrode into the amygdala is used to kindle thebrain, inducing epileptic episodes and other symptoms of epilepsy.Neural stem cell progeny are transplanted into the hippocampal region.The animals are later tested for epileptic episodes and then sacrificedfor analysis of the grafted tissue.

F. ALZHEIMER'S DISEASE

Cognitive impairment is induced in rats and mice by ibotenic acidlesions of the nucleus basalis, or old animals, exhibiting signs ofdementia, are used. Neural stem cell progeny are transplanted into thefrontal cortex, medial septal nucleus and the nucleus of the diagonalband of the brains of the animals. After a survival period, the animalsare tested for cognitive ability and are then sacrificed to allowanalysis of brain tissue.

G. SPINAL CORD INJURY AND DISEASE

Spasticity is a debilitating motor disorder that is a common consequenceof disorders such as spinal cord injury, MS, and cerebral palsy.Transection of the spinal cord is used to produce muscular paralysis andis followed by the development of spasticity, which is characterized bydebilitating hyperactive tendon reflexes, clonus and muscle spasms.Neural stem cell progeny are prepared and are transplanted into thelumbar lateral funiculus. After a survival period, the animals areexamined for improvement in motor control and are then sacrificed toallow for analysis of spinal tissue.

                                      TABLE III                                   __________________________________________________________________________    DONOR CELL       TRANSPLANT       BrdU/                                         SOURCE HOST REGION BrdU BrdU/GFAP NeuN                                      __________________________________________________________________________    Embryonic CD1                                                                          Neonate CD1                                                                           striatum                                                                              +  +     +                                             Mouse Mouse frontal cortex + + +                                               Adult CD1 striatum + + +                                                      Mouse hippocampus + + +                                                        frontal cortex + + +                                                          parietal cortex + + +                                                         MS/NDB + + +                                                                 Adult Wistar Rat spinal cord + + +                                             hippocampus + + +                                                             parietal cortex + +                                                         Adult CD1 Mouse Adult CD1 striatum + +                                         Mouse hippocampus +                                                            frontal cortex +                                                             Adult Wistar Rat spinal cord +                                             __________________________________________________________________________

                  TABLE IV                                                        ______________________________________                                        Donor Cell          Transplant           BrdU/                                  Source HOST Region β-Gal BrdU GFAP                                     ______________________________________                                        Embryonic Adult CD1 hippocampus                                                                              +    +    +                                      CD1 Mouse Mouse frontal cortex + + +                                          in vitro gene  parietal cortex + + +                                          transfer  striatum + + +                                                        MS/NDB + + +                                                                Embryonic Adult DC1 striatum + + +                                            Rosa Mouse parietal cortex + + +                                                MS/NDB + + +                                                                Adult Rosa 26 Adult C57/ hippocampus +                                         BL/6 Mouse frontal cortex +                                                    MS/NDB +                                                                  ______________________________________                                    

                                      TABLE V                                     __________________________________________________________________________    DONOR CELL SOURCE                                                                          HOST           β-Gal                                                                        BrdU/GFAP                                     __________________________________________________________________________    Embryonic Rosa 26 Mouse                                                                    Adult 6-OHDA lesioned                                                                        +   +                                                C57BL/6 mouse                                                                 (striatal injections)                                                         Adult 6-OHDA lesioned Wistar + +                                              rat                                                                           (striatal injections)                                                        Embryonic Rosa 26 Mouse Adult Mutant Weaver Mouse + +                          (striatal injections)                                                      __________________________________________________________________________

All references, patents, and patent applications cited herein areincorporated herein by reference.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 8                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GAGATGCGAC CCTCAGGGAC            - #                  - #                      - # 20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - GTCCCTGAGG GTCGCATCTC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - TAAATAAAAG ATGCCCTGG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - CCAGGGCATC TTTTATTTA             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GAACTGGGAT GTGGGGCTGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - CCAGCCCCAC ATCCCAGTTC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - GCCAGCGGCA TCACCTCG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - CGAGGTGATG CCGCTGGC             - #                  - #                      - #  18                                                                 __________________________________________________________________________

What is claimed is:
 1. A method for inducing in vivo proliferation ofprecursor cells located one in neural tissue of a mammal comprisingadministering a fibroblast growth factor and at least one additionalgrowth factor selected from the group consisting of epidermal growthfactor, transforming growth factor alpha, and amphiregulin to saidmammal to induce in vivo proliferation of at least one precursor cell toproduce precursor cell progeny.
 2. A method of transplanting multipotentneural stem cell progeny into a mammal comprising the steps of:a)administering a composition comprising one or more growth factors tosaid mammal to induce in vivo proliferation of at least one precursorcell to produce precursor cell progeny, wherein said one or more growthfactors induces in vitro proliferation of multipotent neural stem cells,b) removing said precursor cell progeny from said mammal, culturing saidprecursor cell progeny in a culture medium in vitro containing one ormore growth factors to induce in vitro proliferation of multipotentneural stem cells to produce additional precursor cell progeny, and c)implanting said additional precursor cell progeny into said mammal. 3.The method of claim 2 further comprising genetically modifying saidprecursor cell progeny in vitro with genetic material encoding at leastone neurological agent.
 4. The method of claim 1 additionally comprisingadministering genetic material to said mammal to produce geneticallymodified precursor cell progeny, wherein said genetic material encodesat least one neurological agent.
 5. The method of claim 1 whereinepidermal growth factor is administered to said mammal.
 6. The method ofclaim 1 wherein said growth factors are administered to a CNS ventricleof said mammal.
 7. The method of claim 6 wherein said CNS ventricle isthe lateral ventricle of the forebrain.
 8. The method of claim 6 whereinsaid CNS ventricle is the central canal.
 9. The method of claim 1wherein said mammal has damaged or missing glial cells and wherein atleast some of said precursor cell progeny differentiate into glialcells.
 10. The method of claim 1 wherein said mammal has damaged ormissing neurons and wherein at least some of said precursor cell progenydifferentiate into neurons.
 11. The method of claim 1 wherein prior toadministering said composition, said mammal exhibits movement disordersassociated with Parkinson's Disease.